Application for an Extension of the Determination of Nonregulated

Status for Insect

- Resistant, Glufosinate Ammonium

- Tolerant Cotton (08

- 340 -0 1p) :

Transformation Event T303 -3

OECD Unique Identifier B CS

- GH ØØ3 -6

Submitted by :

________________________________ Michael Weeks, M

. S. Registration and Compliance Specialist Regulatory Affairs

– BioScience

Bayer CropScience LP

2 T.W. Alexander Drive

P.O. Box 12014

Research Triangle Park, NC 27709 Telephone: 919

- 549

- 2119

e- mail: michael.weeks@bayer.com

Contributors:

Isabelle Coats Penny Hunst

Mark Rinehardt

January 30

, 2012RELEASE OF INFORMATION

The information in this petition is being submitted by Bayer CropScience LP for review by USDA as part of the regulatory process. By submitting this information, Bayer CropScience LP does not authorize its release to any third party except to the extent it is requested under the Freedom of Information Act (FOIA), 5 U.S.C., Section 552 and 7 CFR 1, covering all or some of this information. Except in accordance with FOIA, Bayer CropScience LP does not authorize the release, publication or other distribution of this information without Bayer CropScience’s prior notice and consent.

©20 11 Bayer CropScience. All rights reserved.

This document is property of Bayer C ropScience and is for use by the regulatory authority to which it has been submitted, and only in support of actions requested by Bayer CropScience. All data and information herein must not be used, reproduced or disclosed to third parties without the written consent of Bayer CropScienceCERTIFICATION

The undersigned certifies, that to the best knowledge and belief of the undersigned, this petition includes all information and views on which to base a determination, and that it includes relevant data and information known to the petitioner which is unfavorable to the petition.

Michael Weeks, M

. S. Registration and Compliance Specialist Regulatory Affairs

– BioScience

Bayer CropScience LP

2 T.W. Alexander Drive

P.O. Box 12014 Research Triangle Park, NC 27709

Telephone: 919

- 549

- 211 9

e- mail: michael.weeks@bayer.comSUMMARY

Bayer CropScience requests a determination from USDA APHIS that insect

- resistant, glufosinate ammonium tolerant cotton event T303

- 3 and any progeny derived from crosses of this event with traditional cotton varieties, and any progeny derived from crosses of this event with transgenic cotton varieties that have also received a determination of nonregulated status, no longer be considered regulated articles under 7 CFR Part 340, and that APHIS consider this document as an extension to petition 08

- 340

- 01p

. The subject of petition 08

- 340

- 01p, TwinLinkTM Cotton, received a

determination of non

- regulated status on September 23, 2011.

Gossypium hirsutum transformation event T303

- 3 was produced by Agrobacterium

mediated transformation utilizing vector pTDL004 containing a cry1Ab gene construct, encoding insect -resistance, and the bar gene as a selectable ma rker conf erring tolerance to glufosinate ammonium herbicides. The antecedent organism, cotton event

T304

- 40 as described in petition 08

- 340

- 01p

, was also generated through Agrobacterium

- mediated transformation utilizing a slightly different vector ( pTDL008 )

. Both events produce the same insecticidal crystal protein (ICP) Cry1Ab (expression product of the cry1Ab gene) and PAT protein (expression product of the bar gene)

.

Molecular analysis has demonstrated that event T303

- 3 contains one stably inte grated

intact copy of the cry1Ab gene and bar gene cassette s

. Evaluation of the Cry1Ab protein expressed by event T303

- 3 was found to be comparable to that expressed by

event T304

- 40 as well as that expressed in E. coli for the purposes of protein safety assessment in petition 08

- 340

- 01p.

Agronomic evaluation s demonstrated that no differences in morpholog y , disease or pest susceptibility were observed when compar ed to other cultivated cotton varieties. No adverse effects on beneficial organisms were found. Composition analysis of raw agricultural commodities produced by event T303

- 3 was found to be substantially equivalent to that of its non

- transgenic counterpart.

Based on all analyses performed, event T303

- 3 was foun d to be comparable to the antecedent organism, event T304

- 40.

A comparison of characteristics of events T303

- 3 and T304

- 40 is summarized in T able 1

and is discussed in the appropriate sections of the petition.Table 1. Comparison of events T304

- 40 and T303 -3

Characteristic T303

- 3 T304

- 40 Crop Cotton Cotton Recipient Organism Gossypium hirsutum Gossypium hirsutum

Parent Line Coker 315 Coker 315

Transformation Method Agrobacterium

- mediated Agrobacterium

- mediated

Trait Insect

- resistant, herbicide

tolerant Insect

- resistant, herbicide

tolerant

Gene Products Cry1Ab and PAT Cry1Ab and PAT

Vector pTDL004 pTDL008

Gene/Donor cry1Ab / Bacillus thuringiensis berliner 1715 cry1Ab / Bacillus thuringiensis berliner 1715

Promoter/Donor P35S2/Cauliflower Mosaic Virus promoter Ps7s7/Subterranean Clover Stunt Virus promoter

Terminator/Donor 3’ me1/3’ untranslated region of NADP

- malic enzyme gene of Flavia

bidentis 3’ me1/3’ untranslated region of NADP

- malic enzyme gene of Flavia

bidentis

Gene/Donor Phosphinothricin acetyltransferase ( bar )/ Streptomyces hygroscopicus Phosphinothricin acetyltransferase ( bar )/ Streptomyces hygroscopicus

Promoter/Donor P35S3/Cauliflower Mosaic Virus promoter P35S3/Cauliflower Mosaic

Virus promoter

Terminator/Donor 3’ nos/3’ untranslated region of nopaline synthase gene from T

- DNA of pTiT37 3’ nos/3’ untranslated region of nopaline synthase gene from T

- DNA of pTiT37 Molecular Weight Cry1Ab ~69 kDA ~69 kDAACRONYMS AND SCIENTIFIC TERMS

a.i. Active ingredient

A Acre

ADF Acid D etergent F iber

ANOVA Analysis of V ariance

BC Backcross

BCS Bayer CropScience bp Base pair

Bt Bacillus thuringiensis cry1Ab Insecticidal crystal protein gene from Bacillus thuringiensis Cry1Ab Insecticidal crystal protein expressed from the cry1Ab gene d.w. Dry weight

F 1 , F 2 , etc Breeding generations denoting product of pollinations and subsequent self

- pollinations following the cross of transformation event with conventional germplasm

ELISA Enzyme

- L inked I mmunosorbent A ssay FWS U.S. Fish & Wildlife Service

g Gram

g Earth’s gravitational acceleration (1 g = 3,530,394 cm / minute 2 )

ICP Insecticidal crystal protein

kDa Kilodalton

lb US pound

LB Left border of T

- DNA

μ g Microgram

MQ Milli

- Q water

MW Molecular W eight

NDF Neutral D etergent F iber

ng Nanogram

ORF Open R eading F rame PAT/ bar Phosphinothricin Acetyl

- Transferase as expressed by the bar

gene

pg Picogram

RB Right border of T

- DNA

SD Standard D eviation

T 1 , T 2 , etc Breeding generations denoting self

- pollinations of the T 0 plant (transformation event )

T

- DNA Transfer DNA from Agrobacterium tumefaciens

TwinLink Insect

- resistant, herbicide

- tolerant stacked cotton product (events T304

- 40 x GHB119)

U.S. United StatesTABLE OF CONTENTS

Title Page

.......................................................................................... ... 1

RELEASE OF INFORMATION

............................................................... ... 2

CERTIFICATION

.................................................................................... 3

SUMMARY

............................................................................................ 4

ACRONYMS AND SCIENTIFIC TERMS

................................................... ... 6

TABLE OF CONTENTS

........................................................................... 7

LIST OF TABLES

.................................................................................... 9

LIST OF FIGURES

.................................................................................. 10

I. RATIONALE FOR NONREGULATED STATUS

..................... 11

II. THE COTTON FAMILY

...................................................... . 1 2

III. DEVELOPMENT OF EVENT T303

- 3

..................................... 13

III. A. The Transformation System

................................................. 1 5

III. B. The Parent al Line

............................................................... 1 5

III. C. Breeding History

................................................................ 1 5

III .D. Generations Used for Analyses

............................................. 17

IV. GENETIC MATERIAL USED FOR TRANSFORMATION

........... 18

IV.A

. Construction of Plasmid pTDL004

.......................................... 18

IV.B. Description of Genetic Elements and Regulatory Sequences

...... 19

IV.C. Identity and Source of Genetic Material

................................... 19

V. GENETIC CHARACTERIZATION OF EVENT T303

- 3

... ............ 20

V.A. Insert Characterization

........................................................ 20

V.A.1. Southern Blot Analyses

....................................................... 20

V.A.2. DNA Sequencing

............................................................... 2 4

V.B. Open Reading Frames and Associated Regulatory Regions

....... 2 5

V.C. Confirmation of the Absence of Vector Backbone

..................... 2 5

V.D. Mendelian Inheritance

......................................................... 3 1

V.E. Conclusions

..................................................................... . 3 1

VI. CHARACTERIZATION OF THE INTRODUCED PROTEINS

... ... 3 2

VI.A. Cry1Ab Protein

.................................................................. 3 2

VI.A.1. History and Background

...................................................... 32

VI.A.2 Characterization of the Cry1Ab Protein

................................... 32

VI.B. The Pat/ bar Protein

............................................................ 3 2

VI.C. Protein Safety

.................................................................... 3 2

VI.D. Expression of Introduced Proteins

.......................................... 33

VI.E. Comparison of Cry 1Ab Protein from Events T303

- 3 and T304

- 40

. 35

VII. AGRONOMIC AND PHENOTYPIC EVALUATION

................... 40

VII.A. History of Field Activities

...................................................... 40

VII.B. Agronomic and Phenotypic Characteristics Evaluated

............... 4 1

VII. B.1. Agronomic and Plant Product Characteristics

........................... 42

VII. B.2. Assessment of Phenotype by Efficacy Against Insect Pests

........ 45

VII. C. Disease and Pest Characteristics

.......................................... 46

VII.D. Composition Analysis

......................................................... 46

VII.D.1. Proximate Analysis

............................................................. 47

VII.D.2. Anti

- Nutrient Analysis

.......................................................... 47

VII.E. Conclusion

........................................................................ 48

VIII. ENVIRONMENTAL SAFETY AND IMPACT ON AGRONOMIC

PRACTICES

....................................................................... 49VIII.A. Potential for Gene Transfer

................................................... 49

VIII.B. Weediness Potential

............................................................ 50

VIII.C. Effects on Non

- Target Organisms

.......................................... 50

VIII.D. Effects on Endangered Species

............................................. 50

VIII.E. Indirect Effects on Other Agricultural Products

.......................... 51

IX. STATEMENT OF GROUNDS UNFAVORABLE

... ..................... 5 2

X. REFERENCES

.................................................................. 5 3

Appendix 1. F ield Trial Termination Reports 2001

- 2006

...... ..................... 57

Appendix 2. Materials and M ethods

.................. .................................... 87

2.A. Insert Characterization

........................................................ 88

2.B. Confirmation of Absence of Vector Backbone

.......................... 89

2.C. Protein ( C ry1Ab and PAT) Expression in Raw Agricultural Commodity

....................................................................... 91

2.D. Protein Characterization

...................................................... 9 2

2.E. Composition Analysis

.......................................................... 9 3

Appendix 3. Characterization of Event T303

- 3

............ ............................ 9 6

3.A. Insert Characterization

......................................................... 9 7

3.B. Confirmation of Absence of Vector Backbone

........................... 10 0

3.C. Composition Analysis

.......................................................... 10 3LIST OF TABLES

1. Comparison of e vents T304

- 40 and T303

- 3

............................................. 5

2. Generations and backgrounds used for analysis

.................................... 17

3

. Genetic E lements of T

- DNA from pTDL004

.............................................. 19

4. Expected and Obtained Hybridization Fragments of Event T303

- 3

............. 21

5. Mendelian segregation of cotton event T303

- 3

....................................... 3 1

6

. Cry1Ab protein levels in event T303

- 3 fuzzy seed

.................................. 34

7

. PAT/ bar protein levels in event T303

- 3 fuzzy seed

................................. 35

8. Field trial locations for evaluation of agronomic and phenotypic

characteristics

................................................................................. 40

9. Agronomic characteristics evaluated in field trials

................................... 41

10. Yield and fiber quality characteristics evaluated in field trials

..................... 41

11. Phenotypic (efficacy) characteristics evaluated in field trials

..................... 41

12. Parameters assessed b y year and location........................................... 42

13. Summary of agronomic data from 2005 field trials

.................................. 42

14. Summary of agronomic data from 2006 field trials

.................................. 43

15. Summary of plant product characteristics from 2005 field trials

.................. 44

16. Summary of plant product characteristics from 2006 field trials

.................. 45

17. Summary of phenotypic characteristics by efficacy against target pests

...... 46

18. Analyses performed on cottonseed of event T303

- 3 and its non

- transgenic counterpart

..................................................................................... 47

19. Mean proximate composition in cottonseed of event T303

- 3

..................... 47

20. Mean anti

- nutrient composition in cottonseed of event T303

- 3

.................. 48

21. Analytical methods used for compositional analysi s

................................ 9 5

22. Event T303

- 3 insert verification

– probe information

................................ 9 7

23. Event T303

- 3 vector backbone analysis

– probe information

..................... 10 0

24. Expected and obtained hybridization fragments with overlapping vector backbone probe templates

....................................... .......................... 10 1

25. Expected and obtained hybridization fragments using T

- DNA probe

........... 10 2

26. Amino acid composition in cottonseed of event T303

- 3

............................ 10 3

27. Fatty acid composition in cottonseed of event T303

- 3

.............................. 10 4

28. Cyclopropenoid fatty acid composition in cottonseed of event T303

- 3

........ 10 4

29. Mineral and tocopherol composition in cottonseed of event T303

- 3

............ 10 5LIST OF FIGURES

1. Schematic of the d evelopment process of event T303

- 3 cotton

................ 14

2. Breeding diagram for event T303

- 3 cotton

........................................... 16

3. Vector map of pTDL004

................................................................... 18

4. Event T303

- 3 insert organization by probe hybridization sizes

................. 2 2

5. Hybridization results utilizing cry1Ab

, bar , and T

- DNA probes

.................. 2 3

6. Insert organization of event T303

- 3

.................................................... 2 4

7. Schematic drawing of probe strategy for vector backbone detection

......... 2 7

8. Event T303

- 3 verification of absence of vector backbone

– Southern blot

results

.......................................................................................... 29

9. Comparison of the Cry1Ab protein from E. coli to the Cry1Ab protein

isolated from events T303

- 3 and T304

- 40

............................................ 36

10. Comparison of trypsin

- digested and thrombin

- engineered Cry1Ab from

E.coli to Cry1Ab isolated from event T304

- 40

....................................... 38

11. Event T303

- 3 insert verification

– Southern blot results

........................... 9 8I. RATIONALE FOR NONREGULATED STATUS

The United States Department of Agriculture (UDSA) Animal and Plant Health Inspection Services (APHIS) is responsible for protection of the US agricultural infrastructure against noxious pests and weeds. Under the Plant Protection Act (7 USC § 7701

- 7772) APHIS considers plants altered or produced by genetic engineering as restricted article s under 7 CFR 340 which cannot be released into the environment without appropriate approvals. APHIS provides that petitions may be filed under 7 CFR §340.6 to evaluate data to determine that a particular regulated article does not present a plant pest r isk

. Should APHIS determine that the submitted article does not present a plant pest risk; the article may be deregulated and released without further restriction s.

This petition serves an application for an Extension of the Petition for Determination of Nonregulated Status for Insect

- Resistant, Glufosinate Ammonium

- Tolerant c otton : TwinLink TM

C otton (events T304

- 40 x GHB119) . The petition for TwinLink Cotton (08

- 340

- 01p) received a determination of non

- regulated status on September 23, 2011.

Event T303

- 3 expresses the same Cry1Ab and PAT/ bar protein s as event T304

- 40 and therefore there are no changes in the rationale from petition 08

- 340

- 01p entitled “Petition for Determination of Nonregulated Status for I nsect- Resistant and Glufosinate

Ammonium

- Tolerant Cotton: TwinLink TM Cotton (events T304

- 40 x GHB119)”.II. THE COTTON FAMILY

The genus Gossypium is classified under the tribe Gossypiae, family Malvaceae, subfamily Malvoideae.

The OEC D consensus document on cotton biology (OECD, 2008 ) provides information pertaining to the following aspects of cotton biology:

• Taxonomy

, morphology and use s

• C enters of origin of the species and domestication

• Agronomic practices

• Reproductive biology and dispersal

• Genetics and hybridization

• In teractions with other organisms

• Human health and biosafetyIII. DEVELOPMENT OF EVENT T303 -3

The T303

- 3 event was obtained using the same transformation system ( Agrobacterium

mediated) as was used to obtain the antecedent organism, event T3 04

- 40 (USDA, 2008) . The parental line used , Coker 315, was the same for event T304

- 40. A schematic of the development process of event T303

- 3 is shown in Figure 1.Figure 1

. Schematic of the development process of event T303

- 3 cotton

c ry1Ab gene (encoding the lepidopteran insecticidal crystal protein Cry1Ab)

bar (encoding the Phosphinothricin Acetyl

- Transferase, PAT)

Assembly of plasmid pTDL004 containing cry1Ab and bar gene cassettes

Agrobacterium

- mediated transfer of T

- DNA from pTDL004

into cotton cells via co- cultivation

Selection of transformation events based on tolerance to glufosinate ammonium herbicide

Regeneration of T 0 cotton plants

Evaluation of transformed plants for agronomic performance

and insect efficacy against target insects

Selfing and continued evaluation of agronomic performance and insect efficacy

Selection of homozygous plants; backcrossing and crossing for product development

Field and laboratory studies to support prod uct registrationIII.A. T he T ransformation System

EventsT304

- 40 and T303 -3 were both generated through Agrobacterium

- mediated gene

transfer of the T

- DNAs from pTDL008 and pT DL004

, respectively. Each T 0 plant was crossed with conventional cotton in order to obtain homozygous and stable lines.

Agrobacterium

- mediated gene transfer of a plasmid results in the transfer of the DNA fragment between the T

- DNA border repeats to the plant genome. The left and right border repeats of A. tumefaciens are also inserted in the individual events. Even though some of the regulatory elements used in the transformation process were derived from

A. tumefaciens , a known plant pathogen, the genes that cause crown gall disease were not part of the T

- DNA , and therefore were not incorporated into the recipient plant (Deblaere et al ., 1985). Section V.C. summarizes the analysis to show the absence of

vector backbone in event T303

- 3.

III.B. The Parental Line

The parental line, Coker 315, is an older commercial variety of upland cotton ( Gossypium hirsutum ) which is no longer commercially cultivated. However, Coker 315 is well suited for transformation due to its capacity for regeneration from tissue culture.

I II. C. Breeding History

The T 0 transformation event of Coker 315 was both self

- pollinated and cross

- pollinated with conventional cotton to produce T 1 and F 1 lines, respectively. The T 1 and F 1 lines

then underwent a series of self
- and cross

- pollinations as well as backcrosses to produce stable lines for the various assessments outlined in this petition. See F igure 2 for a schematic of the crossing scheme utilized.Figure 2. Breeding diagram for event T303

- 3 cottonIII. D. Generation s Used for A nalyses

Table 2 provides the generation and genetic background of event T303

- 3 used for each analyses presented in this petition.

Table 2. Generations and backgrounds used for analysis Analysis Conducted Generation Insert Characterization by Southern Blot Analysis BC 2 F 2

DNA Sequencing BC 2 F 2 ORF Analysis BC 2 F 2 Absence of Vector Backbone BC 2 F 3 Mendelian Inheritance T 1 , T 2 , F 2 , BC 3 F 3 Protein Expression T 4 Comparison of Cry1Ab proteins from

events T303

- 3 and T304

- 40 T 4

2005 Agronomic and Phenotypic

Evaluation in Field T 4

2006 Agronomic and Phenotypic

Evaluation in Field T 5

Composition Analysis T 4IV. GENETIC MATERIAL USED FOR TRANSFORMATION

IV.A. Construction of Plasmid pTDL004

The vector pTDL004 is a derivative of the vector pGSV20 in which the bar gene cassette coding for the phosphinothricin acetyltransferase protein of Streptomyces hygroscopicus (Thompson et al ., 1987) was inserted together with the cry1Ab gene cassette encoding the Cry1Ab crystal protein of Bacillus thuringiensis subsp. berliner (Höfte et al ., 1986). A map of plasmid pTDL004 is provided in F ig ure 3

.

Figure 3. Vector map of pTDL004IV.B. Description of Genetic Elements and Regulatory S equences

Regulatory sequences in the T

- DNA plasmid of vector pTDL004 include two copies of the P35s promoter sequence (P35s3 and P35s2), 3’nos terminator sequence, 3’me1 terminator sequence, and the 5’e1 leader sequence. Genetic elements in the T- DNA plasmid of vector pTDL004 are the bar and cry1Ab ( Cry1Ab5PGS3a ) gene seq uences

. Also included in the T

- DNA of vector pTDL004 are the left and right border repeats (LB and RB, respectively) from the pTDL004 vector. Other genetic elements of vector pTDL004 occupying nucleotide positions from 5176 bp to 13904 bp (ORI pVS1 and O RI colE1) should not be inserted into the plant genome during the transformation process.

For event T303

- 3 cotton, absence of these genetic elements ( i.e., vector backbone) was confirmed (Section V.C.).

IV.C. Identity and Source of G enetic M aterial

Nucleotide position s within the plasmid pTDL004, orientation, identity and source of genetic elements and regulatory sequences are provided in Table 3

.

Table 3. Genetic elements of T

- DNA from pTDL004V. G ENETIC CHARACTERIZATION OF EVENT T303 -3

G enetic charact erization of event T303

- 3 demonstrated that one copy of the cry1Ab gene cassette and one copy of the bar gene cassette were inserted as a result of the

transformation. The DNA sequence of the pre

- insertion locus as well as the insert was determined. The 5’ and 3’ flanking sequence s of the insertion site of event T303

- 3 and the pre

- insertion locus of wild type G. hirsutum were determined and no rearrangements of genomic DNA were found . Further investigation confirmed the absence of any elements of the vector backbone.

V .A Insert Characterization

V.A.1. Southern Blot Analyses Southern blot analyses were used to characterize the insert present in event T303 -3 (Habex et al ., 2007)

. To this end, several i dentical ly prepared membranes were loaded

with samples of event T303 -3 genomic DNA digested with nine different restriction

enzymes

. T he appropriate positive control sa mples (genomic DNA from non

- transgenic cotton supplemented with one copy pTDL004 DNA digested with Not I) and negative

controls (genomic DNA from non

- transgenic cotton) were also prepared and included on

the membranes. The blots were sequentially hybridized with different probe s

, e ither designed to hybridize to a specific component of the cry1Ab or bar gene cassette s or to the complete T

- DNA. A detailed description of the materials and methods employed for

the insert characterization of event T303

- 3 are provided in Appendix 2.A. A summary of

the restriction enzy mes and T

- DNA probes used as well as expected and obtained hybridization fragments is provided in Table 4.Table 4 . Expected and Obtained Hybridization Fragments of Event T303 -3Based on the obtained hybridization results, the organization of the inserted transgenic sequences of event T303

- 3 were determined and demonstrated that the insert of event

T303

- 3 has one complete copy of each the cry1Ab and bar gene cassettes

. F igure 4 shows a schematic presentation of the insert organization deduced by hybridization fragment sizes . R estriction enzymes and probes as well as the expected hybridization fragments are indicated

.

Figure 4

. Event T303

- 3 insert organization by probe hybridization sizes

Figure 5 shows the results of the Southern analyses utilizing the cry1Ab

, bar , and complete T

- DNA probes. Additional results for all Southern analyses utilized for insert characterization are presented in Appendix 3.A.Figure 5. Hybridization results utilizing **cry1Ab**

, **bar** and T

- DNA probes

**cry1Ab** probe **bar** probe

Complete T

- DNA probe

Loading Sequence

- All Probes Shown

Lane 2: G. hirsutum event T303

- 3
– Bam HI digested

Lane 3: G. hirsutum event T303

- 3
– Bgl II digested

Lane 4: G. hirsutum event T303

- 3
– Sac I digested

Lane 5: G. hirsutum event T303

- 3
– Kpn I digested

Lane 6: G. hirsutum event T303

- 3
– Nco I digested

Lane 7: G. hirsutum event T303

- 3
– Pvu I digested

Lane 8: G. hirsutum event T303

- 3
– Xba I digested

Lane 9: G. hirsutum event T303

- 3
– Eco RV digested

Lane 10: G. hirsutum event T303

- 3
– Not I digested

Lane 11: G. hirsutum wild type
– Not I digested

Lane 12: G. hirsutum wild type + 1 copy pTDL004

– Not I digestedV.A.2. DNA Sequencing

DNA sequencing of the transgenic locus confirmed the results of the characterization by Southern analyses which indicated that the inserted sequence of event T303

- 3 has one intact copy of each the cry1Ab and bar gene cassette s (Moens et al., 2007)

. DNA sequencing also confirmed the arrangement of the inserted DNA elements as deduced from Southern analyses ( Figure 6 ). As shown below , the insertion resulted in rearrangements of genetic elements of the T- DNA from pTDL004. However, each of the gene cassettes were inserted as complete an d single copies

Figure 6 . Insert organization of event T303

- 3The pre

- insertion locus, the inserted sequences and the flanking sequences were determined by DNA sequence analys i s of event T303 -3 and non

- transgenic G. hirsutum (Moens et al ., 2007). The results showed a target site deletion of 1184 bp. The putative function of the deleted sequence was not explored; however agronomic and composition analys e s (Section V II ) indicate that event T303

- 3 is comparable to othe r G. hirsutum

varieties.

Event T303

- 3 flanking sequences of 776 bp at the 5’ and 251 bp at the 3’ junctions

, respectively, were determined and were shown to be identical to sequences at the pre

insertion locus of non

- transformed G. hirsutum

.

V.B . O pen Reading Frames and Associated Regulatory Regions

During the transformation process, the T

- DNA from plasmid pTDL004 underwent some rearrangements creating several new junctions as shown in Figure 6

. For these regions, an investigation was conducted as to whether any novel unexpected proteins could be expressed due to the insertion and rearrangement of the gene cassettes (De Pestel, 2008)

.

Open reading frame (ORF) analysis and gene search tools were applied to predict the presence of any potential newly created coding sequences in the junction regions. Seven newly created ORFs were identified and characterized. Bioinformatics tools were applied to look for regulatory elements needed for transcriptio n and translation of any potential new ORFs

. Four putative promoter regions were identified , however, none of them were found to be in a configuration likely to initiate transcription of the predicted

ORFs. Thus , it was concluded that the expression of a new protein s from the seven predicted ORFs was highly unlikely as the regulatory elements necessary to initiate transcription and translation were not present.

In addition, the putative amino acid sequences of the seven ORFs were also compared with seque nces of known allergens and toxins using publically available protein databases Unipro t- Swissprot (release 55.6, 2008) , Uniprot

- TrEMBL (release 37.8, 2008) , PDB (release 2007) , DAD (release 37.8, 2007)

, and GenePept (release 165,

2008 ) ( Capt, 2009 ). Searches included epitope homology search using overlapping eight amino acid sequences and overall homology search utilizing BLASTP algorithm. The matching criteria was 100% identity match over a linear contiguous eight amino acid segment and 35% identity match over the full

- length query sequence with a known toxin or allergen for epitope and overall homology searches , respectively. No significant similarities were found between the putative seven ORF sequences and any sequences

from the databases.

The se findings show that the ORF analysis of the insertion present in event T303 -3 did not identify any potential for new proteins to be expressed and moreover, putative amino acid sequence s d id not show homology to a ny known toxin s or allergen s.

V.C

. Confirmation of the Absence of Vector Backbone

Southern analysis, confirmed the absence of vector backbone in event T303 -3 (Habex, 2006). Samples of g enomic DNA from event T303 -3

, non

- transgenic control plants (negative control), and DNA from plasmid pTDL004 (positive control) were analyzed using seven overlapping vect or backbone probes covering the vector backbonesequences of plasmid pTDL004. Detailed materials and methods are provided in Appendix 2.B. A sc hematic diagram illustrating the probe strategy is shown in Figure 7

.Figure 7. Schematic drawing of probe strategy for vector backbone detectionExpected and obtained hybridization fragments by probe , test material, and digest used are provided in Appendix 3 Table 24 (vector backbone probes) and 25 (T

- DNA probe)

.

Genomic DNA from event T303

- 3 plants and negative control plants showed no hybridization with any of the vector backbone probes. Hybridization occurred with the positive control, demonstrating the conditions were appropriate for hybridization to occur (Figure 8 ).Figure 8. Event T303

- 3 verification of absence of vector b ackbon e – Southern blot

r esults

PT002

- 1 probe PT003

- 1 probe

PT005

- 1 probe PT007

- 1 probe

Loading Sequence All Probes

Lane 2: G. hirsutum event T303

- 3
– Eco RV digested

Lane 3: G. hirsutum event T303

- 3
– Not I digested

Lane 4 : G. hirsutum wild type
– Not I digested

Lane 5: G. hirsutum wild type
– Not I digested + 0.1 copy pTDL004
– Not I digested

Lane 6: G. hirsutum wild type
– Not I digested + 1 copy pTDL004
– Not I digested

Lane 7: G. hirsutum wild type

– Not I digested + 10 copy pTDL004

– Not I digestedFigure 8 (continued). Event T303

- 3 verification of absence of vector backbone – Southern blot r esults

PT008

- 2 probe PT009

- 1 probe

PT010

- 1 probe PT011

- 1 probe

Loading Sequence All Probes

Lane 2: G. hirsutum event T303

- 3
– Eco RV digested

Lane 3: G. hirsutum event T303

- 3
– Not I digested

Lane 4 : G. hirsutum wild type
– Not I digested

Lane 5: G. hirsutum wild type
– Not I digested + 0.1 copy pTDL004
– Not I digested

Lane 6: G. hirsutum wild type
– Not I digested + 1 copy pTDL004
– Not I digested

Lane 7: G. hirsutum wild type

– Not I digested + 10 copy pTDL004

– Not I digestedV.D. Mendel ian Inheritance

The Mendelian segregation of event T303

- 3 was analyzed, using chi

- square analysis, at several stages of introgression of event T303

- 3 into conventional lines as well as in several

inbred lines of the T 0 transformant

. To determine whether event T303

- 3 was segregating as expected each generation was sprayed with glufosinate ammonium herbicide to

eliminate herbicide

- susceptible plants. The results shown in Table 5 illustrate that event

T303

- 3 behaves as a single dominant allele at one locus, with the expected segregation ratio of 3:1 and is stably integrated within and across generations

.

Table 5. Mendelian segregation of cotton event T303 -3

Generation Ratio Observed Expected Chi

Square

T c S d T c S d

T 1 3:1 40 9 37 12 1.1496 e

T 2 3:1 23 6 22 7 0.2873 e

F 2 a 3:1 16 6 15 5 2.5 f

BC 3 F 3 b 3:1 18 6 15 9 0.3 f

a Self- pollinated hemizygous plant.

b Hemizygous BC 3 F 2 plant, self

- pollinated.

c Tolerant to glu fosinate herbicide, positive segregant

d Susceptible to glu f osinate herbicide, null segregant

e The c hi

- square value at the 95% level is 3.84. To be significantly different, the calculated chi

- square value would need to be greater than 3.84.

f The chi

- square value at the 95% level is 5.99. To be significantly different, the calculated chi

- square value would need to be greater than 5.99.

V.E

. Conclusions

Results of the molecular characterization of event T303

- 3 indicate that the event contains a single insert containing the cry1Ab and bar gene expression cassette s

. The results

demonstrated that the inserted DNA contains the cry1Ab and bar genes and their respective regulatory elements in a functional order. The insert has been shown to be stably inherited in several different genetic backgrounds and generations using Mendelian segregation analysis that tracked phenotypic tolerance to glufosinate ammonium herbicide

. Those results clearly indicate that the trait is inherited as expected for a single locus. Southern blot analysis also confirmed the absence of the transformation vector backbone.

G enomic flanking sequences at the 5’ and 3’ junctions of the insertion in event T303 -3 were sequenced and found to match those of the pre

- insertion locus. Bioinformatics analysis of ORFs did not identify any homologies with known allergen and toxin

sequences.

With the exception of the promoter for the cry1Ab gene, event T303

- 3 cotton contains the same genetic eleme nts as the antecedent organism. Genetic characterization

demonstrates that event T303

- 3 is no more likely to become a plant pest in the

environment than the antecedent organism, event T304

- 40.VI. CHARACTERIZATION OF THE INTRODUCED PROTE INS

Event T303

- 3 and the antecedent organism

, event T304

- 40 , were transformed to produce the same insecticidal crystal protein (ICP) Cry1Ab coded for by the cry1Ab gene isolated

from Bacillus thuringiensis berliner and the same PAT protein expressed by the bar gene from Streptomyces hygroscopicus

. Event T304

- 40 is one of the parental events in TwinLink

C otton (T304

- 40 x GHB119) and as such has been previously reviewed by USDA APHIS as part of the determination of non

- regulated status ( USDA

- APHIS, 2011 ).

VI .A Cry1Ab Protein

VI .A.1 History and Background

There are no changes in the history and background of the Cry1Ab protein from the previous petition 08

- 340

- 01p entitled “Petition for Determination of Nonregulated Status for

Insect

- Resistant and Glufosinate

- Ammonium

- Tolerant Cotton: TwinLink TM Cotton (events

T304

- 40 x GHB119)” (USDA, 2008)

.

VI .A.2 Characterization of the Cry1Ab Protein

Petition 08

- 340

- 01p demonstrated the Cry1Ab protein produced in planta by event T304

- 40 was comparable to the Cry1Ab protein isolated from E. coli (USDA, 2008)

. For the protein safety data provided in the petition to also be applicable to the Cry1Ab protein produced by

event T303

- 3, a dditional analysis by SDS

- PAGE was performed to demonstrate that the Cry1Ab protein produced in planta by event T303 -3 is comparable to tha t produced by

event T304

- 40 and the E. coli

- derived protein.

Because the Cry1Ab protein in T303

- 3 cotton was shown to be comparable to that of event

T304

- 40 (Section VI.E.), n o additional information on the characterization beyond that presented in petition 08

- 340

- 01p is provided

.

VI .B The PAT/ **bar** Protein

PAT protein biochemistry and mode of action are included in the peer

- reviewed scientific journal published by Herouet et al

. (2005) and in US

- FDA BNF 000086 (US

- FDA, 2003). Briefly, phosphinothricin (L

- PPT) and demethylphosphinothricin (DMPT) are inhibitors of glutamine synthetase. This inhibition results in the accumulation of toxic ammonium ions

and a decrease in the amount of glutamine, an essential amino acid used in many anabolic processes. The PAT enzyme is an acetyltransferase that specifically cataly zes the acetylation of both L

- PPT and DMPT. Enzymatic properties of the PAT protein are well

characterized, in particular, pH and temperature dependency are well

- described and

under stood. From the perspective of safety, this characterization demonstrates that metabolic effects of the expression of the PAT protein are limited to conferring tolerance to the herbicide glufosinate ammonium.

VI .C Protein Safety

Since the Cry1Ab and PAT proteins expressed in T303

- 3 cotton are the same as expressed in T304

- 40 cotton, no additional safety information is available that was not presented in petition 08

- 340

- 01p. In summary, in order to assess any potential adverse

effects to humans or animals resulting from environmental release of the crops expressingthe Cry1Ab and PAT proteins, Bayer CropScience (BCS) has conducted a detailed safety evaluation based on Codex Alimentarius Commission (Codex Alimentari us, 20 03). As a basis, BCS performed a series of safety studies with these proteins, including homology searches of the amino acid sequences with comparison to all known allergens and toxins from large public databases, an in vitro digestibility assay of the proteins, and an acute toxicity test in the mouse. Moreover, publicly available review documents issued by regulatory authorities, indicating that similar protein family members are safe, have been used for supporting this safety assessment. The results of studies conducted by BCS are

consisten t with the published information, confirming that the crops containing these proteins can be safely used as food or feed.

Lack of allergenic potential

• The Cry1Ab and PAT proteins have no amino acid sequence similarity to known allergens, as demonstrated by overall amino acid and epitope homology searches;

• As expected, the Cry1Ab and PAT proteins only have similarities with other Cry or PAT proteins;

• The Cry1Ab and PAT proteins do not share epitopes with known allergens;

• The Cry1Ab and PAT proteins are not glycosylated;

• The Cry1Ab and PAT proteins are not heat stable;

• The Cry1Ab and PAT proteins are degraded by human simulated gastric and intestinal fluids. This minimizes the likelihood that these proteins could survive in the human digestive tract and be a bsorbed.

Lack of toxic potential

• The Cry1Ab and PAT proteins have no amino acid sequence similarity to known allergens, as demonstrated by overall amino acid homology searches;

• The Cry1Ab and PAT proteins are degraded by human simulated gastric and intestinal fluids. This minimizes the likelihood that these proteins could survive in the human digestive tract and be absorbed;

• There were no mortalities, clinical signs or treatment

- related effects in female OF1 mice after an acute oral administration by gavage of the Cry1Ab, Cry2Ae or PAT proteins at 2,000 mg protein/kg body weight.

In conclusion, it is considered that the cry 1Ab and bar genes as well as the Cry1Ab and PAT proteins are not toxic to mammals and do not possess any of the characteristics associated with food allergens. Therefore, no effects on animal and human health are to be expected by consumption of the cry1Ab or bar genes and the Cry1Ab or PAT proteins.

VI. D. Expression of Introduced Proteins

The content of the Cry1Ab and PAT proteins in ginned cottonseed (also referred to as fuzzy seed) of event T303

- 3 was determined by enzyme

- linked immunosorbent assays (ELISA) specific to each protein ( Robinson, 2008 ). Detailed methods for analysis of expression levels of Cry1Ab in raw agricultural commodity are provided in Appendix 2.C.

Since fuzzy seed is not delinted, it could not be ground into a homogeneous material. A procedure was developed previously to effec tively remove the lint coat from the kernel, creating two fractions (kernel and lint coat). These two fractions were analyzed separately for Cry1Ab and PAT content and then added together to re

- create the amount in the original fuzzy seed. Seed was colle cted from event T303

- 3 plants grown in six field trials inthe U.S. One unsprayed event T303

- 3 sample and three event T303

- 3 samples sprayed

with glufosinate ammonium from each of the sites were analyzed. Unsprayed, non

transgenic seed (control) from each of the trial sites was also analyzed.

On a dry weight basis, the average Cry1Ab protein content from all test sites of cotton fuzzy seed samples from unsprayed event T303

- 3 plants ranged from 7.14 μ g/g to 17.9 μg/g ( overall average value = 11.7 ± 3.74 μ g/g) and from 10.9 μ g/g to 19.1 μ g/g in fuzzy seed samples from sprayed event T303

- 3 plants (overall average value = 14.3 ± 3.50 μ g/g) (Table 6 ). Cry1Ab protein constitutes 0.005073% of the total crude protein in unsprayed transgenic fuzzy seed and 0.005862% in sprayed transgenic fuzzy seed. No Cry1Ab protein was detected in the non

- transgenic control fuzzy seed samples. As a comparison,

the Cry 1Ab content of event T304

- 40 in unsprayed fuzzy seed was 1.29 + 0.52 μ g/g d.w. (0.000561% of total crude protein) and 1.54 + 0.318 μ g/g d.w. (0.000561% of total crude protein). These difference s in the amount of Cry1Ab protein does not affect the efficacy displayed toward the target pest organisms and the Cry1Ab protein represents a small component of the total protein in events T303

- 3 and T304

- 40.

Table 6

. Cry1Ab protein levels in event T303

- 3 fuzzy seed

Sample Average Cry1Ab Content (μg/g Sample) Average Cry1Ab Content (as % of total crude protein)

No Glufosinate Glufosinate Sprayed No Glufosinate Glufosinate Sprayed
Fuzzy Seed 17.9 17.3 0.007668 0.007442

11.4 15.4 0.005758 0.006873

7.14 10.9 0.003004 0.004631

11.3 11.8 0.004450 0.004436

9.06 11.3 0.003901 0.004255

13.7 19.1 0.005659 0.007537
Range in

Values 7.14
– 17.9 10.9
– 19.1 0.003004

– 0.007668 0.004255

– 0.007537

Average ± SD 11.7 ± 3.74 14.3 ± 3.50 0.005073 ±

0.001648 0.005862 ±

0.001578

On a dry weight basis, the average PAT protein content from all test sites of transgenic cotton ranged from 56.5 μg /g to 130 μg /g d.w. in unsprayed fuzzy seed (overall average

value = 102 + 26.9 μg /g) and from 89.3 μg /g to 142 μg /g in sprayed fuzzy seed (overall average value = 118 + 20.7 μg /g) (Table 7 ) . PAT protein constitutes 0.043618% of the total crude protein in unsprayed transgenic fuzzy seed and 0.048027% of the total crude protein in sprayed transgenic fuzzy seed. No PA T/ bar protein was detected in the non

transgenic unsprayed fuzzy seed. As a comparison, the PAT/ bar protein of event T304

- 40

was 163 + 32 μg /g (0.0728% of total crude protein) in unsprayed transgenic fuzzy seed

and 163 + 27 μg /g (0.0736% of total crude protein) in sprayed transgenic fuzzy seed.

These differences in the amount of the PAT/ bar protein do not affect the level of tolerance to the herbicide between the two events and PAT protein represents a small component of the total protein content of the two events.Tab le 7

. PAT/ **bar** p rotein levels in event T303

- 3 fuzzy seed

Sample Average PAT/ bar Content (μg/g Sample) ± SD Average PAT/ bar Content (as % of crude protein) ± SD

No Glufosinate Glufosinate Sprayed No Glufosinate Glufosinate Sprayed
Fuzzy Seed 113 115 0.048430 0.049645

82.7 101 0.041649 0.045095

56.5 89.3 0.023766 0.038042

114 142 0.044902 0.053390

130 125 0.055968 0.047537

113 138 0.046993 0.054451
Range in

Values 56.5
– 130 89.3
– 142 0.023766

– 0.055968 0.038042

– 0.054451

Average ± SD 102 ± 26.9 118 ± 20.7 0.043618

± 0.010832 0.048027

± 0.006020

VI .E Comparison of Cry1Ab Proteins from E vents T303

- 3 and T304

- 40

Safety assessment studies, such as the acute toxicity study, require large amounts of the target protein. The expression of the Cry1Ab protein in event T303

- 3 cotton tissues is relatively low and thus, purification of sufficient quantities of the protein for safety

assessment studies from cotton tissue is not feasible . Thus, Cry1Ab protein was produced using E. coli . The protein produced by the bacterium was engineere d to match the amino acid sequence of the Cry1Ab protein produced in planta. A full protein characterization study (SDS

- PAGE, western analysis, glycol

- staining, LC/MS, N

- terminal sequencing) has been conducted on the Cry1Ab protein, expressed by event T30 4- 40, in the combined event produ ct TwinLink (T304

- 40 x GHB119 ) ( USDA , 2008 ). Therefore, a SDS

- PAGE analysis was conducted to compare the Cry1Ab protein isolated from event T303

- 3 cotton plants to the Cry1Ab protein isolated from event T304

- 40 cotton plants and the microbially

produced Cry1Ab from E. coli (Haas, 2009)

. A summary of the methods is provided in Appendix 2.D

.

The Cry1Ab protein isolated from event T303

- 3, event T 304

- 40 and E. coli was analyzed by SDS

- PAGE to compare their respective molecular weight. Figure 9 shows the Coomassie stained gel . The Cry 1Ab from events T303

- 3 and T304

- 40 have equivalent

measured mobilities of 25 mm. The mobility of the trypsin digested Cry1Ab from E. coli (26mm) is greater than that of the plant

- derived Cry1Ab proteins due to the loss of 28 amino acids which were cleaved during the production of the protein.Figure 9

. Comparison of the Cry1Ab protein from **E. coli** to the Cry1Ab protein isolated from events T303

- 3 and T304

- 40The C oomassie stained gel in Figure 10 shows a comparison of the electrophoretic mobility of trypsin digested Cry1Ab protein from E. coli and a thrombin

- engineered Cry1Ab protein

from E. coli to the Cry1Ab isolated from event T304

- 40 cotton leaves. The thrombin

engineered Cry1Ab protein is representative of the plant form of Cry1Ab expressed in

events T303

- 3 and T304

- 40 cotton and is converted to the active form when ingested by the target in sect. The measured mobility of this protein and the Cry1Ab protein from event

T304

- 40 cotton leaves was 32mm. The measured mobility of the trypsin

- digested Cry1Ab was 34mm. Plotting of the electrophoretic mobility vs . the respective molecular weight (MW ) yielded the approximate molecular weight. For the Cry1Ab protein from the T30 4- 40 cotton leaves and the thrombin protein standard, the approximate MW was 68.2 kDa. The approximate MW for the trypsin

- digested protein was 62.6 kDa.Figure 10

. Comparison of trypsin

- digested and thrombin

- engineered Cry1Ab from **E. coli** to Cry1Ab isolated from event T304

- 40In summary, the SDS

- PAGE results indicate that the E. coli

- produced Cry1Ab proteins are comparable to the Cry1Ab protein s isolated from events T303

- 3 and T304

- 40. Therefore, protein characterization and safety data for the Cry1Ab produced by event T304

- 40 provided in petition 08

- 340

- 01p adequately addresses the same criteria for event T303

- 3.VI I. AGRONOMIC AND PHENOT YPIC EVALUATION

Agronomic and phenotypic characteristics of event T303

- 3 were evaluated in field trials in multiple locations in typical cotton growing regions of the US in 2005 and 2006

. Field trials were conducted according to common agricultural practices for a given growing region.

In addition, all field trials of T303

- 3 conducted from 2001 to 2006 for increasing research seed as well as agronomic and phenotypic evaluation were observed for unusual or unexpected characteristics in regards to susceptibility to plant disease, weediness potential, effect on non

- target organis ms

, beneficial insects, and the overall environment (Appendix 1)

.

VI I .A History of Field Activities

Fi eld trials for characterization of agronomic and plant product quality (i.e., fiber quality parameters) were conducted in 2005 under acknowledged notification 05

- 040

- 06n. In 2006 field trials were conducted under acknowledged notification 06

- 047

- 02n to assess agronomics, plant product quality, and pheno typic characteristics (i.e., efficacy). A summary of field trial locations for the assessment of agronomics, plant product quality, and phenotypic characteristics is provided in T able 8

. Locations for agronomic and phenotypic evaluation were representati ve of major portions of the US Cotton growing regions including the Texas High Plains and Gulf Coast

, and the Louisiana Delta region.

Table 8

. Field trial locations for evaluation of agronomic and phenotypic

characteristics



### Notification

Number Year Number of

Locations Locations

05

- 040

- 06n 2005 3 Gaines Co., TX Uvalde Co., TX Wharton Co., TX

06

- 047

- 02n 2006 5 Bossier Co., LA Gaines Co., TX Lubbock Co., TX Wharton Co., TX (2) a

a Two sites at this location

Field trials conducted in 2005 for agronomic and plant product quality assessment included homozygous event T303 -3 of Coker 315 (T 4 generation) compared with wild type Coker

315.

In 2 006

, comparisons for agronomics and plant product quality were assessed in four field trials. Phenotype (i.e., efficacy) parameters were assessed in up to five field trials; however fewer field trials were utilized for the assessment of certain parameters

. Field

trials in 2006 utilized homozygous event T303

- 3 in the Coker 315 background (T 5 generation) and wild type Coker 315 as the comparator. In each of these trials, entries of homozygous event T303

- 3 an d the comparator were infested or not infested with larva of Helicoverpa zea Boddie (cotton bollworm). Plots which were not artificially infested wer e sprayed with conventional insecticides according to university extension recommendations

if natural infestations were observed.Similar field trials were used for assessment of the antecedent organism, event T304

- 40, in

2007 and 2008.

VI I.B Agronomic and Phenotypic Characteristics Evaluated

A summary and description s of agronomic , yield and plant product quality, and phenotypic characteristics evaluated in 2005 and 2006 field trials are provided in Table 9, 10, and 11

.

Table 9

. Agronomic characteristics evaluated in field t rials

Parameter Description Emergence Evaluation of the germination rates and plant population

Height Average plant height from cotyledonary node to terminal, expressed in inches

Total number of nodes Number of reproductive nodes present on the main stem of the plant Height to node ratio (HNR) Plant height divided by total number of nodes

Table 10. Yield and fiber quality characteristics evaluated in field trials

Parameter Description

Yield Productivity expressed as pounds of lint produced per acre

Gin t urnout Lint weight divided by seed cotton weight, expressed as a percentage Seeds per boll Average number of seeds per boll

Seed index Average weight in grams of 100 seed, an indication of seed

size and maturity. Fiber length Average length of the longer one

- half of cotton fibers

Fiber strength The force in grams required to break a bundle of fibers one tex unit in size (1 tex = weight in grams of 1,000 meters of fiber)

Fiber m icronaire A measure of fiber fineness and maturity as indicated by specific surface area

Fiber uniformity % Ratio between the mean length and upper half mean length of the fibers expressed as a percentage

Table 11. Phenotypic (efficacy) characteristics evaluated in f ield t rials

Parameter Description

Flower s

- % damaged A measure of the percent open flowers damaged by insects

Flowers

- living larvae A count of living insect larva present in 25 flowers Square s

- % damage d A measure of the percent immature flowers (squares) damaged by insects Squares

- living larvae A count of living insect larva present in 25 squares

Boll s

- % damage d A measure of the percentage of fruit (bolls) damaged by

insects

Bolls

- living larvae A count of living insect larva present in 25 bollsAgronomic, plant product quality, and phenotype parameters were not assessed at a ll locations each year. Table 12 provides a matrix of which parameters were assessed at

each field trial location.

Table 12. Parameters assessed by year and location

Location County/ST YearEmergenceHeightTotal nodesHNRYieldGin turnoutSeeds perbollSeed indexFiber lengthFiber strengthFibermicronaireFiberuniformityFlowers-% damagedFlowersliving larvaeSquares-% damagedSquaresliving larvaeBolls-%damagedBolls-living larvae

Gaines/TX ‘05 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Uvalde/TX ‘05 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Wharton/TX ‘05 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Bossier/LA ‘06 ■ ■ ■ ■ Gaines/TX ‘06 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Lubbock/TX ‘06 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Wharton/TX ‘06 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Wharton/TX ‘06 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■

VII.B.1 Agronomic and Plant Product Characteristics

In 2005, s ome small differences were observed in agronomic characteristics between homozygous event T303

- 3 cotton and the comparator

. In most cases, event T303

- 3 cotton was within the ranges observed for TwinLink Cotton (the product of the breeding cross of

event GHB119 and event T304

- 40, the antecedent organism). A summary of agronomic

data from 2005 field trials across lo cations is pr esented in Table 13 along with range of means for TwinLink Cotton from 2007 and 2008 field trials for the same parameters

.

Table 13 . Summary of a gronomic data from 2005 f ield t rials

Parameter Coker 315

WT

Mean Coker 315

T303

- 3

Mean Standard

Deviation TwinLink Range of Means a Emergence

plants / ft (3) b 0.44 0.23 0.098 1.36
– 3.57

Height

inches (3) 38.81 41.16 2.210 30.33
– 38.17

Total Nodes (3) 22.34 24.67 0.712 16.95
– 23.00

HNR

(3) 1.74 1.67 0.040 1.62
– 3.74

a Range of mean s for TwinLink Cotton (GHB119 x T304

- 40) from petition 08

- 340

- 01p

b Number of field trials where parameter was measured WT = Wild type HNR = Height to node ratio

General ly, event T303

- 3 Coker 315 and wild type Coker 315 were comparable for most parameters and means were within one standard deviation of each other. Further, agronomic parameters for event T303

- 3 fell within the range of values observed for TwinLink Cotton, or at most approximately one standard deviation outside that of TwinLinkCotton. Comparisons of TwinLink Cotton to event T303

- 3 should be made with the consideration that the data does not come from the same field trials or the same year and

therefore variation due to weather and location is expected.

In 2005, emergence was particularly low for both wild type Coker 315 and event T303

- 3. This was due to poor seed quality as a result of the delinting process for preparing planting seed. The process for preparing planting seed was improved upon for the 2006 field trials as evidenced by markedly improved emergence for both wild type Coker 315 and event

T303

- 3 cotton. In 2006 field trials, all agronomic parameters evaluated were in line with

the observed values for TwinLink Cotton from 2007 and 2008 field trials (Table 14).

Table 14. Summary of agronomic data from 2006 field trials

Parameter Coker 315 WT (I) Coker 315

WT Coker 315

T303

- 3 (I) Coker 315

T303

- 3 Standard

Deviation TwinLink Range of Means a Emergence

plants / ft (4) b 3.53 3.65 3.07 3.49 0.473 1.36
– 3.57

Height

inches (4) 38.46 37.43 38.66 38.02 1.196 30.33
– 38.17

Total Nodes (4) 20.67 19.87 21.33 20.88 0.518 16.95
– 23.00

HNR

(4) 1.84 1.87 1.80 1.82 0.077 1.62
– 3.74

a Range of means for TwinLink Cotton (GHB119 x T304

- 40) from petition 08

- 340

- 01p Appendix 4

b Number of field trials where parameter was measured WT = Wild type (I) = Infested with H. zea HNR = Height to node ratio

Plant product characteristics were generally comparable among event T303

- 3 and wild type Coker 315 in 2005 field trials (Table 15). All plant product characteristics of event

T303

- 3, with the exception of micronaire, were within the range of means observed for TwinLink Cotton in 2007 and 2008 field trials. In the case of micronaire, event T303

- 3 was within one standard deviation of that observed for TwinLink Cotton. Seed per boll was not evaluated in TwinLink Cotton field trials and therefore is not prese nted in Table 15.Table 15. Summary of plant product characteristics from 2005 field trials

Parameter Coker 315

WT

Mean Coker 315

T303 -3

Mean Standard

Deviation TwinLink Range of Means a

Yield

lb / A

(3) b 1180.56 749.22 68.657 608.00
– 1434.00

Gin Turnout

% Lint (3) 39.74 38.91 0.619 35.97
– 45.40

Seed per Boll (3) 11.06 12.00 0.534
- - -

Fiber Length

1/32 nd inch (3) 1.16 1.25 0.001 1.11
– 1.30

Fiber Strength g / TEX (3) 29.20 30.98 0.869 30.05
– 34.77

Micronaire (3) 4.09 3.69 0.249 3.77
– 5.25

Fiber Uniformity

% (3) 83.06 85.60 0.403 81.75
– 87.87

a Range of mean s for TwinLink Cotton (GHB119 x T3 04

- 40) from petition 08

- 340

- 01p

b Number of field trials where parameter was measured WT = Wild type HNR = Height to node ratio

Again in 2006 field trials, plant product characteristics were generally comparable between wild type Coker 315 and event T303

- 3 in both the infested and non

- infested treatments (Table 16). Means for event T303

- 3 from either treatment were generally within the ranges of that observed for TwinLink Cotton. Micronaire, fiber strength and yield were within one standard deviation of that observed for TwinLink Cotton. Again seed per boll and seed

index were not evaluated in TwinLink Cotton field trials.Table 16

. Summary of plant product characteristics from 2006 field trials

Parameter Coker 315 WT (I) Coker 315

WT Coker 315

T303

- 3 (I) Coker 315

T303

- 3 Standard

Deviation TwinLink Range of Means a

Yield

lb / A

(4) b 756.19 942.08 584.36 613.00 73.404 608.00
– 1434.00

Gin Turnout

% Lint (4) 38.03 38.29 37.62 37.37 1.092 35.97
– 45.40

Seed per Boll (4) 30.90 32.70 22.42 22.71 1.202
- - -

Seed Index g / 100 seed (4) 7.99 7.69 9.05 8.51 0.749
- - -

Fiber Length

1/32 nd inch (4) 1.17 1.16 1.24 1.25 0.020 1.11
– 1.30

Fiber Strength g / TEX (4) 28.65 28.80 29.33 28.87 0.745 30.05
– 34.77

Micronaire (4) 4.27 4.06 3.56 3.46 0.252 3.77
– 5.25

Fiber Uniformity

% (4) 82.92 82.37 82.84 83.12 0.405 81.75
– 87.87

a Low and high mean for TwinLink Cotton (GHB119 x T304

- 40) from petition 08

- 340

- 01p Appendix 4

b Number of field trials where parameter was measured WT = Wild type (I) = Infested with H. zea

Event T303

- 3, when compared with wild type Coker 315, consistently had lower yields in both 2005 and 2006 field trials. This in part led to the decision to move forward with the antecedent organism, event T304

- 40, as the “Cry1Ab component” in the TwinLink Cotton breeding stack. Aside from yield, event T303

- 3 was comparable to wild type Coker 315

and TwinLink cotton in all other agronomic and plant product characteristics. T hough

event T303

- 3 demonstrated lower yield potential, this does not in itself present any greater

risk of event T303

- 3 becoming a plant pest.

VI I .B. 2 Assessment of Phenotype by Efficacy Against Insect Pests

The efficacy assessments conducted in 2006 field trials showed that the phenotype of

event T303

- 3 was performing as expected and that the Cry1Ab protein expressed in event

T303

- 3 was effective in protecting the plant from cotton bollworm

.

A summary of effic acy data is presented in Table 17 . As expected , event T303

- 3 in either

the infested or non

- infested treatments had lower damage ratings on squares, flowers, or bolls compared to the wild type Coker 315, infested or not infested with cotton bollworm

. The s ame is true in terms of living larvae of cotton bollworm found on squares, flowers, or bolls. For all assessments made, event T303

- 3 plots that were infested or not infested

were similar to each other. These results confirm that the Cry1Ab protein expressed byevent T303

- 3 effectively controls the target insect pest it was challenged with in these field trials and the phenotype of event T303

- 3 is comparable to the antecedent organism.

Table 17 . Summa ry of phenotypic characteristics by efficacy against target pests

Parameter Coker 315 WT (I) Coker 315

WT Coker 315

T303

- 3 (I) Coker 315

T303

- 3 Standard

Deviation

Flowers % Damaged (3) a 8.8 3.8 1.9 1.5 1.984

Flowers Living Larvae (3) 1.73 1.50 0.40 0.20 0.666

Squares % Damaged (5) 19.63 10.86 2.20 1.14 5.716

Squares Living Larvae (4) 3.27 1.77 0.28 0.14 1.708

Bolls % Damaged (5) 5.56 1.97 1.72 0.50 1.477

Bolls Living Larvae (4) 0.34 0.23 0.14 0.06 0.171

a Number of field trials where parameter was measured WT = Wild type (I) = Infested with H. zea

VI I .C Disease and Pest Characteristics

Personnel conducting the field trials also visually monitored plant disease and pest

resistance characteristics of event T303

- 3 cotton and non

- transgenic controls . There were no differences reported in severity of disease symptoms or insect damage (other than the targeted insects susceptible to the Cry1Ab protein) between the transgenic plants and the

non

- transgenic cotton plants . For additional information please see the field trial

t ermination r eports provided in Appendix 1

.

VII.D Composition Analysis

To conduct compositional analysis of event T303

- 3 cotton, six field trials were conducted in Georgia, Arkansas, Mississippi and Texas, all important cotton

- growing regions of the southern U.S. The plants in the trials were grown under conditions typical of cotton production practices. At each site, there were six transgenic plots and three non

transgenic plots. T hree of the transgenic plots at each site were sprayed twice with a tank mix of glufosinate ammonium herbicide at 0.52 lb a.i./A and ammonium sulfate at 3.0 lbs/A (target application rate). The other three transgenic plots at each site were left unsprayed

.

Nine samples of fuzzy seed (ginned cottonseed) were collected from each of the six field trials (Haas, 2007). There were a total of 18 samples from each of three groups: non

transgenic Coker 315 cotton, T303

- 3 cotton that was unsprayed and T303

- 3 cott on thatwas sprayed with glufosinate herbicide. Analyses of the seed samples were carried out at Eurofins Scientific Des Moines, IA (Table 18 ). For list of the analytical methods used refer

to Table 21 in Appendix 2 .E

.

Table 18

. Analyses p erformed on cottonseed of event T303 -3 and its n on

- transgenic

c ounterpart

VII.D.1 Proximate Analysis

Table 19 shows the results of the proximate analysis of event T303

- 3 cottonseed. These results are comparable to the proximate analysis of TwinLink (T304

- 40 x GHB119) as provided in petition 08

- 340

- 01p (USDA, 2008)

.

Table 19

. Mean proximate composition in cottonseed of event T303 -3

Parameter Non

Transgenic Transgenic Not sprayed Transgenic Sprayed Reference

ranges a Mean ± SD Mean ± SD Mean ± SD

Moisture %fw 8.64 ± 1.77 9.42 ± 1.34 9.71 ± 1.91 4.0

– 15.9

Protein %dm 23.01 ± 2.06 24.05 ± 1.70 24.63 ± 1.83 11.7

– 34.2

Fat %dm 17.38 ± 2.90 19.05 ± 1.37 19.13 ± 1.46 11.8

– 36.3

Ash %dm 4.26 ± 0.56 4.53 ± 0.45 4.65 ± 0.57 3.2

– 5.0

Total Carbohydrates %dm b 55.35 ± 3.29 52.37 ± 2.08 51.60 ± 2.47 36.4
– 74.4

ADF %dm 40.58 ± 4.56 39.48 ± 4.20 36.80 ± 3.46 29.0

– 66.9

NDF %dm 50.02 ± 5.90 48.85 ± 4.03 45.80 ± 4.47 38.1

– 71.4

Data represent an average of three replicate samples at six field test sites.

a Reference ranges compiled from OECD (2004) , ILSI (2007) , USCA (1982) , Calhoun et al. (1995) , Bertrand et

al. (2005) , Amann (1999) , Lundquist (1995) , Berberich et al. (1996) and Nida et al. (1996)

. b Total carbohydrates calculated as 100%
- (protein %dm + fat %dm + ash %dm)

Additional composition data on amino acids, fatty acids, minerals and vitamins is provided in Appendix 3.C. Literature values for the individual analyses of cottonseed are also presented for reference. Values for event T303

- 3, in either the sprayed or unsprayed treatment, were comparable with those the non

- transgenic counterpart and within the reference ranges from the literature.

VII.D.2 Anti

- Nutrient Analysis

Fuzzy seed of event T303

- 3 was also analyzed for the content of anti

- nutrients of cotton

— gossypol and phytic acid ( Haas, 2007). Th e r esults are presented in Table 20

. The values for gossypol and phytic acid were within the reported literature ranges and were similar for

the non

- transgenic, the transgenic unsprayed and transgenic sprayed plants and comparable to that of TwinLink Cotton (USDA, 2008 ). Matrix Analyses Performed
Cottonseed (fuzzy seed) Proximates: moisture, ash, fat, protein, including carbohydrate calculation; acid detergent fiber; neutral detergent fiber

, Antinutrients: g ossypol (free,”

- “, “+” and total); phytic

acidTable 20 . Mean a nti

- nutrient composition in cottonseed of event T303 -3

Parameter Based on dry matter

Reference

ranges a Non

Transgenic Transgenic Not sprayed Transgenic Sprayed

Mean ± SD Mean ± SD Mean ± SD Free gossypol % 0.53 ± 0.12 0.56 ± 0.06 0.53 ± 0.06 0.23

– 1.40 (−)Gossypol % 0.32 ± 0.07 0.32 ± 0.05 0.32 ± 0.04 0.18

– 0.77 b
(+)Gossypol % 0.41 ± 0.08 0.44 ± 0.04 0.43 ± 0.05 0.28

– 1.22 b
Total gossypol % 0.73 ± 0.15 0.76 ± 0.08 0.75 ± 0.08 0.46

- 1.99 Phytic acid % 1.63 ± 0.29 1.65 ± 0.25 1.70 ± 0.28 0.85

- 2.57

Data represent an average of three replicate samples at six field test sites.

a Reference ranges compiled from OECD (2004) , ILSI (2007)

, Calhoun et al. (1995), Berberich et al. (1996)

, Nida et al. (1996)

. , Phelps et al. (1965), and Wozenski and Woodburn (1975 )

b The proportion of (

- )gossypol and (+) gossypol in whole, fuzzy cottonseed was reported as 38.8% and 61.2% of total gossypol (Calhoun et al., 1995); on the basis of the range for total gossypol of 0.4 6
– 1.99 % dm this equals to 0.18
– 0.77% dm for (

- ) gossypol and 0.28

– 1.22% dm for (+) gossypol.

VII.E. Conclusion

Overall, these evaluations indicate that event T303

- 3 does not show any unexpected changes in plant morphology as compared to conventional cotton

. Any statistical ly significant differences observed are unrelated to the introduced trait. The agronomic performance data indicate no biologically meaningful differences between even t T303

- 3

and the non

- transgenic counterpart nor the antecedent organism, event T304

- 40

. No

differences between the e vent T303 -3 and the non

- transgenic control were observed for disease occurrence or severity and response to insect pressure, with the except ion of the target pests.

Samples of fuzzy seed of event T303 -3 were analyzed for their proximate , amino acids, fatty acids, minerals, vitamins and anti

- nutrient composition and compared to a non

transgenic control. Published data ranges were provided as additional points of reference

. Plant material was collected from six locations and analyzed using standards met hods

. The ranges and standard deviations overlapped for event T303 -3 and the non

- transgenic

control and fell within the publis h ed data ranges.VII I. ENVIRONMENTAL SAFETY AND IMPACT ON AGRONOMIC PRACTIC ES

VI I I.A

. Potential for Gene Transfer

O nly two wild Gossypium species are present in the US: G. thurberi Todaro found in mountain regions of Arizona at altitudes of 2500 to 5000 feet and G. tomentosum which is found in Hawaii. Only G. tomentosum is capable of crossing with domesticated cotton that will produce fertile offspring. There is no expected selective advan tage conferred by th e introduced cry1Ab and bar genes in event T303 -3 if that cross would occur.

Vertical Gene Flow:

Cotton pollination Gossypium hirsutum is considered to be a self

- pollinating crop. Cotton pollen is heavy and sticky thus cross pollination by wind is unlikely. Cotton can, however, be pollinated by insects. Honeybees ( Apis mellifera ) and bumblebees ( Bombus spp.) are the primary insect pollinators. McGregor (1976) traced the movement of pollen from a cotton field surrounded by a large number of honeybee colonies. Movement of the pollen was traced by means of fluorescent particles. McGregor found that at 150 to 200 feet away from the source plant, only 1.6 percent showed the presence of the fluorescent particles. By comparison, the isolation distances for Foundation, Registered and Certified seeds in 7 CFR Part 201 are 1320, 1320 and 660 feet, respectively. The trend for cross pollination to decrease as the distance from the source increased has been established by several research groups over the years

. (Kareiva et al

., 1994; Van Deynze et al., 2005).

Outcrossing potential to wild/weedy relatives The potential for outcrossing can be defined as the ability of gene escape to wild cotton relatives. Previously

, the USDA stated in the environmental assessment document of LLCotton25 cotton that “gene flow from genetically engineered cotton into wild cotton

relatives are not likely, and if it occurs, would not lead to increased wee diness” (USDA, 2003). G. tomentosum, found only in Hawaii, is the only species capable of crossing with domesticated cotton that will produce fertile offspring. Outcrossing to G. tomentosum is unlikely as there is no cotton production in Hawaii other than winter nursery breeding

a ctivities where isolation practices are employed, and therefore the potential for gene flow

to these wild relatives is low. There are other wild relatives known to exist in Southern Florida and Puerto Rico that are capable of crossing with cultivated cotton. However, these wild relatives are found hundreds of miles from where cotton production occurs.

Outcrossing potential to feral or cultivated cotton No feral cotton populations (domesticated plants capable of surviving outside of cultivation)

of G. barba dense have been found in the US Cotton production area. Seed production fields (production of planting seed) are required to be isolated from other cotton fields to prevent cross pollination. Therefore if any cross pollination were to occur to either G.

b arbadense or G. hirsutum it would be from a lint production field where seed is crushed and not propagated.

Potential for Horizontal Gene Flow: Bayer CropScience is not aware of any reports of incidents of naturally occurring transgene

movement from transgenic crops to sexually incompatible species.VI I I.B Weediness Potential

In the United States, cotton ( G. hirsutum ) is not a weed pest and has no sexually compatible weedy relatives except perhaps G. tomentosum in Hawaii where there is no

commerci al cotton production. A number of references confirm the lack of weediness of

cotton such as Crockett ( 1977 ) , Holm et al. ( 1979 )

, and Muenscher ( 1980 ) . The USDA has previously determined that “cotton is not considered to be a serious, principal or common weed pest in the US” (USDA, 2003). Previous findings by the USDA of similar herbicide

tolerant cotton during environmental assessment expected no change in weediness potential , and an example is glufosinate ammonium tolerant cotton (LibertyLink),

commercia lly sold today. The largest concern is that of volunteer plants that could become weedy in subsequent years. Volunteers are also limited by the geography in which they may exist as cotton does not survive as a perennial where freezing temperatures are

reached during the winter. Volunteers can easily be controlled by crop rotation, tillage and/or pre
- or post

- emergence herbicides. For example, glufosinate ammonium tolerant cotton volunteers could easily be controlled by using the herbicide glyphosate.

There is l imited probability that event T303 -3 cotton or any Gossypium speci es containing

event T303 -3 cotton would become a weed problem. The data presented in this petition

did not indicate any significant differences in phenotypic or plant morphological

cha racteristics between event T303 -3 cotton and the conventional non

- transgenic comparator line that would impact plant pest or noxious weed potential. Based on this data

there is no e vidence to suggest that T303 -3 cotton has a higher likelihood to become a

p lant pest than conventional cotton or event T304

- 40 cotton which has been granted non

regulated status as part of the de regulation of Twin Link C otton on September 23, 2011 ( USDA , 2011 ) . There were no instances in which volunteer monitoring after harvest revealed any differences in survival or persistence relative to other cotton varieties.

VII I .C Effects on Non

- Target Organisms

An assessment of the risk to non

- target species has been performed for the Cry1Ab protein expressed in T304

- 40 cotton. Since the Cry1Ab protein in T303

- 3 cotton is the same protein, the assessment for T304

- 40 informs the assessment of T303

- 3. The toxicity of the Cry1Ab protein expressed in T303

- 3 cotton is expected to be similar. Testing with microbially

- derive d Cry1Ab protein at levels greatly exceeding the expressions levels found in Cry1Ab transgenic plants resulted in no effects on several beneficial species ( USDA,

2008 ). Therefore, the data presented in the previous petition adequately addresses toxicity

to humans and non

- target organisms for event T303

- 3 plants.

VI I I.D Effects on Endangered Species

The responsibility for endangered species falls to t he US Fish & Wildlife Services (FW S) under the Endangered Species Act (ESA) (16 USC §1531). Section 6 of the ESA requires federal agencies who conduct activities which may affect listed species to consult with the FWS to ensure that listed species are protected should there be a potential imp act.

It is not anticipated that the use of event T303

- 3 cotton will impact any current ly listed species of concern. Species of concern that may inhabit areas close to commercial cotton operations would not be add it ionally impacted by the use of T303

- 3 cotton. Commercial agriculture routinely disturbs the ground in which crops are currently planted. As a result,perennial vegetative species would not grow in these areas. Additionally, because horizontal gene flow to sexually incompatible species is not an issue, there is negligible potential for exposure to the transgene contained in event T303

- 3 cotton through sexual reproduction.

Cry1Ab protein is highly specific to lepidoptera, so the only organisms which might be

consid ered to be ‘at risk’ from T303

- 3 cotton would be endangered lepidopteran insects. While there are endangered lepidopteran species in cotton growing counties ( e.g. Kern Primrose Sphinx moth, Saint Francis' Satyr butterfly) the larvae are highly unlikely to be exposed to Cry1Ab protein because their habitats do not overlap with cotton fields and their larvae do not feed on cotton. The amount of pollen that would drift from these cotton plants onto plants fed upon by endangered or threatened species would be v ery small. It is also therefore highly unlikely that event T303

- 3 cotton could outcross to any wild or weedy

relatives of cotton.

For these reasons, it is not believed that the use of T303

- 3 cotton in commercial cotton production will adversely impact endangered species of concern.

VI I I.E Indirect Effects on Other Agricultural Products

T303

- 3 cotton is an alternative cotton product that contains insect

- resistant and herbicide

tolerant traits. Current agricultural practi ces already in use for these types of products are not expected to change with the introduction of event T303

- 3 cotton

. Adoption and use of genetically modified cotton can provide positive impacts on agricultural practices. These positive impacts have been detailed in a study by Brook es and Barfoot (2008 ) and include:

Herbicide

- tolerant crops • Increased management flexibility that comes from a combination of the ease of use

associated with broad

- spectrum, post

- emergent herbicides; • Compared to conventional crops, where post

- emergent herbicide application may result

in ‘knock

- back’ (some risk of crop damage from the herbicide) ; this problem is less likely to

occur in herbicide

- tolerant crops; • Facilitation of adoption of no/reduced tillage practices with resultant savings in time and equipment usage; • Improved weed control has reduced harvesting costs
– cleaner crops have resulted in reduced times for harvesting; • Elimination of potential damage caused by soil

- incorporated residual herbicides in follow

on crops.

Insect

- resistant cr ops • Production risk management/insurance purposes

– taking away the worry of significant pest damage occurring; • A ‘convenience’ benefit (less time spent on crop walking and/or applying insecticides); • Savings in energy use

– mainly associated with less spraying; • Savings in machinery use (for spraying and possibly reduced harvesting times); • Improved health and safety for farmers and farm workers (from reduced handling and use of insecticides);

Genetically modified herbicide

- tolerant cotton was first grown commercially in the US in

1996 and by 201 0 , was planted on 73 % of the total cotton plantings (USDA ERS, 2011)

.IX. S TATEMENT OF GROUNDS UNFAVORABLE

Bayer CropScience know s of no study data and/or observations associated with insect

resistant herbicide

- tolerant event T303

- 3 cotton that will result in adverse environmental consequences for its introduction. TwinLink Cotton (T304

- 40 x GHB119)

, which contains

the antecedent e ve nt T304

- 40

, received nonregulated status in September 2011 ( USDA

, 2011 ). S ince the Cry1Ab and PAT protein s in event T303

- 3 cotton are the same as those

of the antecedent event , the assessment for T304

- 40 informs the assessment of T303

- 3.

The evidence and data provided in this petition supports the conclusion that event T303

- 3 cotton presents a low risk to human health and the environment and does not pose a plant pest risk.X. R EFERENCES

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and R.L. Fuchs. 1996. The composition o f insect

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Bertrand, J.A., T.Q. Sudduth, A. Condon, T.C. Jenkins, and C. Calhoun. 2005. Nutrient

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of the Agrobacterium tumefaciens plasmid pTiAch5. The EMBO Journal 3:835

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- 3 and T304

- 40 cotton, Gossypium hirsutum , USA, 2009. Bayer CropScience Internal Report. 20p.

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transgenic c ounterpart, USA, 2005. Bayer CropScience Internal Report. 130p.

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R.J. van der Klis, and D. Rouan. 2005. Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate

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. Petition 02

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- 01p to USDA/APHIS seeking

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. 2011. Determination of Nonregulated Status for TwinLink TM C otton ( Gossypium

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the File BNF No. 000086. Glufosinate tolerant cotton transformation event LLCotton25. June 5,

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Wozenski J., and M. Woodburn. 1975. Phytic acid (myoinositol hexaphosphate) and phytase activity in four cottonseed protein products. Cereal Chemistry 52:665

- 669.Appendix 1. F ield Trial Termination Reports 2001

- 2006

### USDA 2001 Termination Report for Bt Co tt on

B ay er Cr o pScience L P

T ri a ls Cond uc t ed: S tat e (Co un ty )

01

- 075

- 08 n : MS (Wa sh i ng to n)

01

- 075

- 18 n : MS (Wa sh i ng to n)

01

- 292

- 01 n : PR (Juana Diaz)

T ri a ls Not Co nduc t ed: S tat e (Co un ty )

01

- 292

- 01 n : PR ( Sa bana Grand e)

01

- 312

- 05 n : PR (Juana Diaz, Sa bana Gra nd e)

Plant ing D at es

May 25

, 20 01 (Wa sh i ng ton C o. , M S) th ro ug h Dec emb er 5

, 200 1 (Juana Diaz, PR)

Ha rvest a nd Plot De st ruc tion D at es

Novemb er 8

, 200 1 (Wa sh ington C o. , M S) th rou gh June 28

, 200 2 (Juana Diaz, PR)

P urp o se

Field tr ials were condu cted to te st the eff ica cy as w ell as br ee ding of tran sg en ic he rb ici de-to le ran t c o tt on

. Tra nsg en ic plan ts con ta in ed the ba r gene ex pr e ss ing the PAT enz ym e, wh ich c on fers resistan ce to th e
bro ad-sp ect ru m he rb ici de gl ufo sin at e-a mm onium, as well as the Cr y 1A b gene all owing resistan ce to attack by vari ou s le pi dop te r an pest sp ecie s.

Obs er vat io ns

Exper ie n ced perso nn el qu al if ied in co tt on cu lt iv ati on perf or m ed all plot ob serv ati o ns

. R ec ord ed

ob serv ati ons for tra nsg en ic and non-transgen ic c on t ro l plo ts were pro vid ed f ro m first s qu are th ro ugh open bo ll growth stages.

P la nt emergen ce p atte rn s were un iform and vig oro us with in bo th plo t t ypes. Ge rmin ati on r ates ra ng ed from

8 0 to 90% (Wa sh ington C o.

, MS).

No m orp holog ical differences were no ted between transgen ic a nd n on -transgen ic p la nts

. The only in-f ield phenotyp ic differen ce ob se rv ed betw een the two genotyp es w as th eir resp ect ive levels of to le ran ce to

le pi dop te r an f ee ding pr essur e.

I ns ect pest sp eci es li s ted for the Wa sh ington C o. , MS s ite in cl ud ed bo llworm ( Helic o ve rpa zea ), p la ntbu gs ( Ly gu s li neolaris ), an d cutoworm s ( Ag ro tis up s il on ). At the Juana Diaz, PR s ite only bo llwor m ( Helic o ve rp a zea ) was re por te d. There were no difference s r ec ord ed in eit h er diversity or de ns it y of ins ect
pest sp ecies fo un d betw een transgen ic and non-transgen ic plots.

A sing le sp eci es of benefic ial i ns ec t, the la dybug ( Hi ppo da mia c on ve rgens )

, was r ec ord ed at th e
Wa sh i ng ton C o. , MS s ite. At Juana Diaz, PR honeyb ees ( Ap is melife ra ) and aph id li on s ( Chrys op a s p la rv ae) were r ec ord ed

. As w it h t he pest sp ecie s

, no differen ces were no ted between sp ec i es pro f iles for th e two plot types.

No phty op at hol og y w as r ec ord ed at eit h er s ite.Post

- Trail Monitoring

Volunteer monitoring revealed post

- season plants at the Washington Co., MS sites with less than ten plants

found on April 17, 2002, and the Juana Diaz, PR site with less than ten plants found on July 24, 2002.

These were hand

- weeded. Clean inspections were established by May 3, 2002 and August 23, 2002, respectively.Field T e r m i n ati o n R e por t

USDA I D# 02

- 058

- 02n P a g e 1 o f 3 

## USDA Fie ld Termin ation Re port

No tification No .: 02-058-02n

A pp li ca nt No .: BT-2A-Cotton- MR

P er mi ttee : Av e ntis CropScience ( Now Bayer CropScience LP )

Res earc h Triang le P ar k, NC; 919-549-2655

Regu la ted A rt icle: H er bic id e-tol era nt, g lu fosinate-a mmonium -tol era nt;

I ns ec t- re sist ant, re sist a nt to Lepidopt era n pe st sp ec ies,
Cotton ( Goss ypium hirsutum )

Site Release Informat io n: As shown below, one (1) of two (2) sit e s w as pla nt ed:

C o un ty / Pa ri s h/Di s t rict S tat e o r T erri to ry Rele a s e S tat us

W a sh i ng t o n M S P la n ted

L u bbo ck T X N o t P la n ted



### Rel ea se in formation for Washington Co., MS is a s
follows:

Acre ag e
Plant ed D at e
Plant ed G er m in at ion D at a Transg enic vs

. Non-transg enic D at e

T er m in at ed

0.3 5 5/ 20 /02 % Em er g ence/ S eedling Vi go r
70 % vs 85 % on 5/ 27 /02

I ni tial S ta nd Co unt P ercen tag e
75 % vs 90 % on 6/4/ 02 11 / 26 / 02

F in al S ta nd Co unt

1. 4 p la n ts p er f oo t vs 1. 6 p la n ts

p er f oo t on 6/ 28 / 02



### Purpose of Release: The purpose of the re l ea se was to te st the eff i cac y of the

t ra nsgenic h er bic id e-tol era nt cotton plants. T ra nsge nic plants conta in ed the Cry 1Ab gene, a llowing re sist an ce

to a tt ac k by v ar ious lepidopt era n pe st sp ec ie s. The

p erf ormance of the t ra nsgenic cotton with re sp ec t to the

nont ra nsgenic count erp ar t plant wa s also eva lu ated, a s

w er e the ov era ll agronomic ch arac t er isti c s.

Obs er vat io ns: The te st site wa s insp ec ted twelve (12) tim es dur ing the

gr owing s ea son (5/27/02, 5/31/02, 6/4/02, 6/19/02,
6/20/02, 6/26/02, 6/28/02, 7/8/02, 7/12/02, 8/1/02, 8/9/02, 8/12/02) for agronomic growth ch arac t er isti cs
and dis ea se and ins ec t p e st in fe st ation.Field T e r m i n ati o n R e por t

USDA I D# 02

- 058

- 02n P a g e 2 o f 3 

### Obs er vations w er e rec orded fr om fir st squ ar e th rough open boll growth stages on both the t ra nsgenic and

nont ra nsgenic plants. T ra nsgenic plants did not

g er min a te quite a s w e ll as the nont ra nsge nic

count erp ar t, but both g re w vi gorously.

Two (2) sp ec ies of ins ec t p e sts w er e note d: th rips and plant bug s. Th rips w er e s ee n on 5/31/02, and pla nt bu g s

w er e obs er ved on 7/12/02 and 8/1/02. Damage leve ls

ra nged fr om sli g ht to mod era te. No di ffere n ce s w ere

rec orded in the div er sity of ins ec t p e st sp ec ie s found

be tw ee n the t ra nsgenic and nont ra nsgenic count er p ar t.

The only ben ef icia l ins ec t obs er ved was the lady bug

whi ch wa s s ee n on 6/19/02, 7/12/02 and 8/12/02.

No dis ea se sus ce ptibility was noted on the t ra nsgenic or

non-t ra nsgenic plants during a ny of the visits m ade on
5/27/02, 6/20/02, 7/8/02 or 11/26/02.

Results: The only in -field phenotypic di ffere n ce s obs er ved

be tw ee n the t ra nsgenic and non-t ra nsgenic plants w er e
in the level of tol era nce to l epidopt era n fee ding p re ssu r e
and tol era nce to h er bic ide (g lu fosinate-a mmonium)

t rea tm ent.

P la nt Dis posit io n : The cr op w a s h ar ve st e d 11/26/02. F ollowing h ar ve st,

a ll re maining pla nt m at er ia l w a s sh re dde d, bu rne d and

disked und er

.

Vol un teer Monitoring: The plot are a wa s visu a lly insp ec ted for volunt ee r cotton plants four (4) tim es during the following

gr owing s ea son. No volunt ee r plants w er e obs er ved during a ny of the visits.

W

ee dine ss Characte risti c s: No di ffere nce in w ee dine ss ch arac t er isti c s b e tw ee n the

t ra nsgenic and non-t ra nsgenic c otton lin e s w as
obs er ved.

Non-Targe t Organisms: No adv er se effec t on non -t ar ge t o rga nisms fr om e ith e r
the t ra nsgenic or nont ra nsgenic pla nts w a s obs er ve d in

the tria l.

Weathe r S y nopsis: W ea th er nota tions indi ca te the site exp er ien ce d no rm al

c lim a tic c onditions du r ing the gr owing s ea son, but w a s

usua lly we t a t h ar ve st tim e.Field T e r m i n ati o n R e por t

USDA I D# 02

- 058

- 02n P a g e 3 o f 3 

### Containment Me asure s: T ra nsgenic cotton plants w er e sep ara ted fr om
conventiona l cotton pla nts by a distance of ~250 fee t. A 40-foot wide p er im eter of nont ra nsgenic cotton plants

bord ere d the te st plot to p re vent the flow of pollen fr om

t ra nsgenic to nont ra nsge nic plants. The border r ows

w er e de st royed a t the conclusion of the tria l and w ere
monito re d a s p ar t of the te st plot the following gr owing

s ea son.Field T e r m i n ati o n R e por t

USDA I D# 02

- 078

- 01n P a g e 1 o f 3 

## USDA Fie ld Termin ation Re port

No tification No .: 02-078-01n

A pp li ca nt No .: BT-2B-Cotton- MR

P er mi ttee : Av e ntis CropScience ( Now Bayer CropScience LP )

Res earc h Triang le P ar k, NC; 919-549-2655

Regu la ted A rt icle: H er bic id e-tol era nt, g lu fosinate-a mmonium -tol era nt;

I ns ec t- re sist ant, re sist a nt to Lepidopt era n pe st sp ec ies,
Cotton ( Goss ypium hirsutum )

Site Release Informat io n: Rel ea se in formation for Washington Co., MS is a s
follows:

Acre ag e
Plant ed D at e
Plant ed G er m in at ion D at a Transg enic vs

. Non-trans ge nic D at e

T er m in at ed

0.3 5 5/ 20 /02 % Em er g ence/ S eedling Vi go r
70 % vs 85 % on 5/ 27 /02

I ni tial S ta nd Co unt P ercen tag e
75 % vs 90 % on 6/4/ 02 11 / 26 / 02

F in al S ta nd Co unt

1. 4 p la n ts p er f oo t vs 1. 6 p la n ts

p er f oo t on 6/ 28 / 02



### Purpose of Release: The purpose of the re l ea se was to te st the eff i cac y of the

t ra nsgenic h er bic id e-tol era nt cotton plants. T ra nsge nic plants conta in ed the Cry 1Ab gene, a llowing re sist an ce

to a tt ac k by v ar ious lepidopt era n pe st sp ec ie s. The

p erf ormance of the t ra nsgenic cotton with re sp ec t to the

nont ra nsgenic count erp ar t plant wa s also eva lu ated, a s

w er e the ov era ll agronomic ch arac t er isti c s.

Obs er vat io ns: The te st site wa s insp ec ted twelve (12) tim es dur ing the

gr owing s ea son (5/27/02, 5/31/02, 6/4/02, 6/19/02,
6/20/02, 6/26/02, 6/28/02, 7/8/02, 7/12/02, 8/1/02, 8/9/02, 8/12/02) for agronomic growth ch arac t er isti cs
and dis ea se and ins ec t p e st in fe st ation.Field T e r m i n ati o n R e por t

USDA I D# 02

- 078

- 01n P a g e 2 o f 3 

### Obs er vations w er e rec orded fr om fir st squ ar e th rough open boll growth stages on both the t ra nsgenic and

nont ra nsgenic plants. T ra nsgenic plants did not

g er min a te quite a s w e ll as the nont ra nsgenic plants, but both g re w vi gorously.

Two (2) sp ec ies of ins ec t p e sts w er e note d: th rips and plant bug s. Th rips w er e s ee n on 5/31/02, and pla nt bu g s

w er e obs er ved on 7/12/02 and 8/1/02. Damage leve ls

ra nged fr om sli g ht to mod era te. No di ffere n ce s w ere

rec orded in the div er sity of ins ec t p e st sp ec ie s found

be tw ee n the t ra nsgenic and nont ra nsgenic count er p ar t.

The only ben ef icia l ins ec t obs er ved was the lady bug

whi ch wa s s ee n on 6/19/02, 7/12/02 and 8/12/02.

No dis ea se sus ce ptibility was noted on the t ra nsgenic or

non-t ra nsgenic plants during a ny of the visits m ade on
5/27/02, 6/20/02, 7/8/02 or 11/26/02.

Results: The only in -field phenotypic di ffere n ce s obs er ved

be tw ee n the t ra nsgenic and non-t ra nsgenic plants w er e
in the level of tol era nce to l epidopt era n fee ding p re ssu r e
and tol era nce to h er bic ide (g lu fosinate-a mmonium)

t rea tm ent.

P la nt Dis posit io n : The cr op w a s h ar ve st e d 11/26/02. F ollowing h ar ve st,

a ll re maining pla nt m at er ia l w a s sh re dde d, bu rne d a nd

disked und er

.

Vol un teer Monitoring: The plot are a wa s visu a lly insp ec ted for volunt ee r cotton plants four (4) tim es during the following

gr owing s ea son. No volunt ee r plants w er e obs er ved during a ny of the visits.

W

ee dine ss Characte risti c s: No di ffere nce in w ee dine ss ch arac t er isti c s b e tw ee n the

t ra nsgenic and non-t ra nsgenic c otton lin e s w as
obs er ved.

Non-Targe t Organisms: No adv er se effec t on non -t ar ge t o rga nisms fr om e ith e r
the t ra nsgenic or nont ra nsgenic pla nts w a s obs er ve d in

the tria l.

Weathe r S y nopsis: W ea th er nota tions indi ca te the site exp er ien ce d no rm al

c lim a tic c onditions du r ing the gr owing s ea son, but w a s

usua lly we t a t h ar ve st tim e.Field T e r m i n ati o n R e por t

USDA I D# 02

- 078

- 01n P a g e 3 o f 3 

### Containment Me asure s: T ra nsgenic cotton plants w er e sep ara ted fr om
conventiona l cotton pla nts by a distance of ~250 fee t. A 40-foot wide p er im eter of nont ra nsgenic cotton plants

bord ere d the te st plot to p re vent the flow of pollen fr om

t ra nsgenic to nont ra nsge nic plants. The border r ows

w er e de st royed a t the conclusion of the tria l and w ere
monito re d a s p ar t of the te st plot the following gr owing

s ea son.Field T e r m i n ati o n R e por t

USDA I D# 02

- 261

- 25n P a g e 1 o f 3 

## USDA Fie ld Termin ation Re port

No tification No .: 02-261-25n

A pp li ca nt No .: BT-2D-Cotton- MR

P er mi ttee : Bayer CropScience LP

Res earc h Triang le P ar k, NC; 919-549-2655

Regu la ted A rt icle: H er bic id e-tol era nt, g lu fosinate-a mmonium -tol era nt;

I ns ec t- re sist ant, re sist a nt to Lepidopt era n pe st sp ec ies,
Cotton ( Goss ypium hirsutum )

Site Release Informat io n: Rel ea se in formation for Sabana G ra nde Dist rict, PR is

a s follows:

Acre ag e
Plant ed D at e
Plant ed G er m in at ion D at a Transg enic vs

. Non-transg enic D at e

T er m in at ed

0.2 2 11 / 19 /02 % Em er g ence/ S eedling Vi go r

> 50 % vs > 50 % on 11 / 30 /02

I ni tial S ta nd Co unt P ercen tag e

> 80 % vs > 80 % on 12 /6/ 02 4/ 23 / 03

F in al S ta nd Co unt P ercen tag e

40

- 60 % vs > 80 % on 12 / 20 / 02



### Purpose of Release: The purpose of the re l ea se wa s for b ree ding and s eed

in crea se. T ra nsgenic plants conta in ed the Cry 1Ab gene,

a llowing re sist ance to a tt ac k by v ar ious lepidopt era n

pe st sp ec ies. The p erf ormance of the t ra nsgenic cotton

with re sp ec t to the nont ra nsgenic count erp ar t plant wa s
also eva lu ated, a s w er e the ov era ll agronomic

ch arac t er isti c s.

Obs er vat io ns: The te st site wa s insp ec ted fift ee n (15) tim es dur ing the

gr owing s ea son (11/26/02, 11/30/02, 12/6/02, 12/8/03,
12/10/02, 12/17/02, 12/20/02, 1/2/03, 2/15/03, 2/23/03, 3/1/03, 3/10/03, 4/5/03, 4/15/03, 4/16/03) for agronomic growth ch arac t er isti c s and dis ea se and ins ec t p e st

in fe st ation.Field T e r m i n ati o n R e por t

USDA I D# 02

- 261

- 25n P a g e 2 o f 3 

### Obs er vations w er e rec orded fr om fir st squ ar e th rough open boll growth stage s on both the t ra nsgenic and

nont ra nsgenic plants. Both the t ra nsgenic and

nont ra nsgenic plants g er min ated we ll and g re w vigorously.

Four (4) sp ec ies of ins ec t p e sts w er e noted: g ra sshopp er s (12/6/02), l eaf min er s (12/17/02), aphids (12/20/02), and

ar my wo r ms (1/2/03). Damage leve ls ra nged fr om sli ght

to mod era te ly sev ere . No di ffere n ces w er e rec orded in

the div er sity of ins ec t p e st sp ec i es f ound be tw ee n the

t ra nsgenic and nont ra nsge nic count erp ar t.

Two (2) sp ec ies of ben ef icia l ins ec ts w er e obs er ved:

cucumber b ee tle on 12/20/02 and honeyb ee s on 3/1/03.

No dis ea se sus ce ptibility was noted on the t ra nsgenic or

non-t ra nsgenic plants during a ny of the visits m ade on
12/6/02, 2/15/03, 3/10/03 or 4/5/03.

Results: The only in -field phenotypic di ffere n ce s obs er ved

be tw ee n the t ra nsgenic and non-t ra nsgenic plants w er e
in the level of tol era nce to l epidopt era n fee ding p re ssu r e
and tol era nce to h er bic ide (g lu fosinate-a mmonium)

t rea tm ent.

P la nt Dis posit io n: The cr op wa s h ar ve st ed over two (2) days: 4/16/03 and 4/17/03. Following h ar vest, a ll re ma ining plant mat er ia l

wa s m ec hani ca lly c ultiv ated 4/23/03.

Vol un teer Monitoring: The plot are a wa s visu a lly insp ec ted for volunt ee r cotton plants five (5) tim es during the following

gr owing s ea son, until no volunt eer s h ad b ee n obs er ved

for two (2) cons ec utive post -s ea son monitoring visits.

Po st- Se ason Vo lun t eer Mo ni to ring

D at e No. Plants Obs er v ed/S tag e Method of De st ruc tion

4/ 30 /03 >50 p la nts /V2 M ec han ically Cu lti v ated

5/ 14 /03 >50 p la nts /V3 M ec han ically Cu lti v ated

6/4/03 1 1 to 5 0 p la nts /V2 M ec han ically Cu lti v ated

6/ 24 /03 None Field M ec han ically Cu lti v ated

7/ 18 /03 None Field M ec han ically Cu lti v atedField T e r m i n ati o n R e por t

USDA I D# 02

- 261

- 25n P a g e 3 o f 3 

### W

ee dine ss Characte risti c s: Th er e wa s no evidence of change in ch arac t er isti c s th at

would enhance su rviva l of the g lu fosinate-ammoniumtol era nt t ra nsgenic cotton plants a s comp are d to the

non-t ra nsgenic cotton plants. No di ffere nce in

w ee dine ss ch arac t er isti c s b e tw ee n the t ra nsgenic and

non-t ra nsgenic c otton lin e s w as obs er ved.

Non-Targe t Organisms: No adv er se effec t on non -t ar ge t o rga nisms fr om e ith e r
the t ra nsgenic or nont ra nsgenic pla nts w a s obs er ve d in

the tria l.

Weathe r S y nopsis: W ea th er nota tions indi ca te the site exp er ien ce d t ypi cal

c lim a tic conditions dur ing the gr owing s ea son.

Containment Me asure s: The Sabana G ra nde te st site is a 59- acr e far m. The site produ ce s no comm erc ia l cr ops. A 40-foot- wide

p er im eter of nont ra nsgenic cotton su rr ounded the te st plot to p re vent the flow of pollen. At tria l conclusion,

the border r ows w er e not h ar ve st ed but w er e de st royed along with the re mn ants of the test plot. The borde r

are a wa s monito re d (a s p ar t of the te st plot) f or
volunt ee r cotton pla nts du ring the p er iod tha t followed.Field T e r m i n ati o n R e por t

USDA I D# 03

- 070

- 12n P a g e 1 o f 3 

## USDA Fie ld Termin ation Re port

No tification No .: 03-070-12n

A pp li ca nt No .: B T/HT -3A-Cotton- MR

P er mi ttee : Bayer CropScience LP

Res earc h Triang le P ar k, NC; 919-549-2655

Regu la ted A rt icle: H er bic id e-tol era nt, Gluf osin ate-a mmonium -tol er a nt;

I ns ec t- re sist ant, re sist a nt to Lepidopt era n ins ec t p e sts, Cotton ( Goss ypium hirsutum )

Site Release Informat io n: All re l ea se sit e s a utho rized under this notifi ca tion w ere planted. Be low ar e acrea ge amounts, planting date s and

t er min ation date s for eac h sit e:

C o un ty /S tate Acre ag e
P l a n t ed D at e P l a n t ed D at e
T er m in at ed

O k ti bb e h a/ M S 2.7 5 5 / 27 / 0 3 10 / 24 / 03

W a sh i ng t o n / M S 1. 2 5 / 29 / 0 3 12 / 5 / 0 3

L u bbo c k / T X 0.6 9 5 / 26 / 0 3 & 6 / 9 / 0 3 1 / 10 / 04



### Purpose of Release: The tria ls w er e e st a blish ed to eva lu a te the eff i cac y of the

h er bic id e-tol era nt/ins ec t- re sist ant cotton plants.

T ra nsgenic plants conta in ed the cry1Ab gene, a llowing

re sist ance to a tt ac k by v ar ious lepidopt era n pe st sp ec ies.

The p erf ormance of the t ra nsgenic cotton with re sp ec t to

the nont ra nsgenic count er p ar t w a s also eva lu ated, as

w er e the ov era ll agronomic ch arac t er isti c s.

Obs er vat io ns: The te st sit e s w er e visua lly insp ec te d multiple tim e s during the gr owing s ea son for agronomic growth

ch arac t er isti c s and dis ea se and ins ec t p e st in fe st ation.

Obs er vations w er e rec orded for the t ra nsgenic and

nont ra nsgenic plants fr om em er gence th rough h ar ve st.

Co un ty or Di st ric t /

S tat e or T erri to ry G er m in at ion D at a Transg enic vs. Non-transg enic

Ok ti bb eh a/ MS M ean germin ati on ra ng ed f ro m 55 % to

95.25 % b et w een the various co tt on li nes.

Wa sh i ng ton /MS % Em er g ence/ S eedling Vi go r
85 % vs

. 85 % on 6/5/03

I ni tial S ta nd Co unt P ercen tag e 90 % vs. 90 % on 6/ 13 / 03

F in al S ta nd Co unt P ercen tag e 90 % vs

. 90 % on 9/ 18 / 03Field T e r m i n ati o n R e por t

USDA I D# 03

- 070

- 12n P a g e 2 o f 3 Co un ty or Di st ric t /

S tat e or T erri to ry G er m in at ion D at a Transg enic vs. Non-transg enic

Lu bbo ck/TX % Em er g ence/ S eedling Vi go r
65.3 % vs

. 62 % on 7/ 16 /03

I ni tial S ta nd Co unt P ercen tag e 65 % vs

. 62 % on 7/ 16 /03

F in al S ta nd Co unt P ercen tag e
65 % vs

. 62 % on 8/ 19 /03



### In Oktibbeha Co., some plots w er e in fe st ed with H. z ea and some w er e not. E ac h w ee k, for a p er iod of six ( 6)

w ee ks, damage and l ar vae counts w er e mad e.

I n gen era l, t ra nsgenic cotton plants exhibited norma l growth and development a t eac h of the lo ca tions.
Exp er im enta l t rea tm e nts of g lu fosinate-a mmonium

w er e made. Ha il d amage n ece ssit ated a re plant in

Lubbock Co.

C o un ty /S tate O b s er vat i o n s /D at es

F un g i/Di s e a s es I n s ect P e s t s B ene f ici a l I n s ec t s

O k ti bb e h a/ M S N o n e ob s e r v ed 6 / 11 / 03

, S o m e p l o ts w e r e i n f e s ted No n o tati o n w as m a d e of

7/6/ 03 , 8/6/03 and w ith H. zea

. No r ec or d the pr esen ce of any

8/ 24 / 03

. made of the pr esen ce of benef icial i ns ect s

.

a n y o t h er i ns ect s p ecie s

. W a sh i ng t o n / M S N o n e ob s e r v ed 6 / 5 / 03

, A li gh t i n f e s tati o n o f p la n t L a d y b eetles w e r e
7/8/ 03 , 7/ 28 /03 and b ug s w as s een 7/ 28 /03 and ob serv ed on 6/5/ 03,

11 / 24 / 03

. 8 / 2 / 03

. 7 / 8 / 0 3 a n d 8 / 2 / 03.

L u bbo c k / T X N o n e ob s e r v ed 7 / 16 / 03

, A li gh t i n f e s tati o n o f L a d y b ug s w e r e ob s e r v ed

7/ 28 /03 and 8/ 15 / 03

. bo ll w or ms and lea fminers 7/ 16 /03 and 8/ 15 / 03,

were ob serv ed 7/ 38 / 03 . adu lt h ood ed b eetles Aphids were ob serv ed were ob serv ed 7/ 28 / 03

, 10 / 20 / 03

. and lace wi ng s were s een
8 / 15 / 03.



### Results: No dis ea se sus ce ptibility or re sist ance di ffere n ce s w ere
obs er ved be tw ee n the t ra nsgenic cotton plants and its

nont ra nsgenic count er p ar t. Some phenoty pic

di ffere n ce s w er e obs er ved be tw ee n the t ra nsgenic and

nont ra nsgenic plants in t er ms of the level of tol era nce to

h er bicide t rea tm e nt and sus ce ptibility to lepidopt era n

pe sts.

P la nt Dis posit io n: The Oktibbeha Co. tria l w a s h ar ve st ed 10/23/03. Stalks

w er e cut and disked under the following day. In Washington Co., h ar ve st o cc u rre d 12/1/03 and 12/2/03.

Stalks w er e mowed, and lint and s ee d burned on

12/5/03. The Lubbock Co. tria l w a s d e st royed 1/10/04 by burning the plot and disking it und er

.Field T e r m i n ati o n R e por t

USDA I D# 03

- 070

- 12n P a g e 3 o f 3 

### Vol un teer Monitoring: The plot area s w er e visua lly insp ec ted for volunt ee r cotton plants during the following gr owing s ea son. The

table below summ ar ize s obs er vations made and ac tions

taken to e limin a te volunt ee r plants.

Po s t

- Se a s o n V o lun t eer M o ni to ring

Co un ty /S tat e D at e No. Plants Method of De st ru ct ion

O b s er v ed/S t ag e

O k ti bb e h a/ M S 5 / 24 / 0 4 N o ne
6/2/04 No ne
7/ 14 /04 No ne
7/ 21 /04 No ne
7 / 28 / 0 4 N o ne

W a sh i ng t o n / M S 4 / 5 / 0 4 N o ne
4/ 13 /04 No ne
4/ 28 /04 No ne
5/6/04 No ne
5 / 14 / 0 4 N o ne

L u bbo c k / T X 4 / 2 / 0 4 N o ne
4/ 16 /04 1 to 1 0 p la n ts P la n ts were remov ed by hand a nd

f ield w as m ec han ically cu lti v ate d.

4/ 30 /04 No ne
5/ 13 /04 1 to 1 0 p la n ts P la n ts were remov ed by hand an d

he rb ici de w as a pp lie d.

5/ 26 /04 No ne

6 / 15 / 0 4 N o ne



### W

ee dine ss Characte risti c s: Th er e was no evidence of change in ch arac t er isti c s th a t

would enhance su rviva l of the t ra nsgenic cotton plants

a s comp are d to the nont ra nsgenic cotton plants. No

di ffere nce in w ee dine ss ch arac t er isti c s w as obs er v ed

be tw ee n the t ra nsgenic and nont ra nsgenic c otton lin e s.

Non-Target Organisms: No adv er se effec t on non-t ar ge t o rga nisms fr om e ith er
the t ra nsgenic or nont ra nsgenic plants was obs er ved during a ny of the tria ls.

Weathe r S ynopsis: The w ea th er in Oktibbe ha Co. was noted to be we t. Conditions for Washington Co. w er e normal. Lubbock

Co. wa s w e t and cold ear ly in the s ea son.

Containment Measure s: A 40 -foot- wide p er im eter of nont ra nsgenic or

comm erc ia l cotton su rr ounded the te st plots to minimi z e pollen flow. The border r ows w er e not h ar ve st e d but

de st royed a t the conclusion of the trial. The bord er

area s w er e monito re d a long with th e ac tu a l t est plot the

nex t growing s ea son for volunt ee r cotton pla nts.

### B T

- 5A

- C o tt

o

n

- MR 1 05

- 0

4

0

- 0

6n US DA Fi e ld T e rmi nat ion R e port

US DA No tifi ca t ion Numb er : 05 -040-0 6n

Appli ca nt In te rn al Numb er : BT- 5A -Co tt

on- MR

Appli ca nt : Ba ye r C ropSci en ce LP

Res ea rch Triang le Park, N C 27 7 09 ( 919 ) 5 49 -26 55

Re gul at ed Ar ticle : Gossypiu m hirsutum; plant incor po ra ted protectant (PIP); toleran t

to

glufosinate-a mm on ium herb ici de

Site Rel ea se In f orm at

io n : Tria ls u tilizi ng th is t ra it were c on du ct

ed at fou r (4 ) l ocati on s. A

fifth site located in Bo ssier Pa r is

h, LA was no t planted:

Coun ty / Sta te A c r ea ge
P l a n t ed D a t e
P l a n t ed D a t e
T e rmin a t ed Is ol at ion Met hod

P i na l C o

., A Z 0

. 4

2

6 5 / 2

7

/ 05 11 / 2

5

/ 05 66 0 ft is o

l a t i o

n d ist an c

e
G a i ne s Co

., TX 0

. 96 5 / 9

/ 05 11 / 7

/ 0

5 4 0 ft is o

l a ti o n b u

ff

er
Uv a l d e C o

. , TX 0

. 2

7 55 4 / 2

2

/ 05 9 / 2

3

/ 05 66 0 ft is o

l a t i o

n d ist an c

e
W

ha r t o

n C o

., TX 0

. 2

3

55 5 / 5

/ 05 10 / 6

/ 05 66 0 ft is o

l a t i o

n d ist an c

e

P urpo se of R el ease : Tria ls w ere estab lis

he d to eval ua te the perfo rm an ce of Pl ant

Incor po rated Protectant trait s in vari ou s cotton varie ti es.

Ob se rvat ion s :

• Pi na l C o

., AZ : Observa ti on s w ere made be tw

een planting and ha rvest fou r ti mes during th e

s

ea son

. A non-tr an s

gen ic c he ck w as no t present to c om pa re the regulated ar ticle to, b ut

c

oo perator ob served no ab no rm a l i ncr ea ses o r decrease s in insec t, dis ea se, o r w ee d pressur e

g iv en the cli mate and re gion the cotton was planted in. C oo perator d id i nd ica te that see d

ge rm i na ti on wa s l ower than no rm a l, but th is was most like ly due to the varie ty

of plant th e

tr

an sgen ic ev en t wa s t ested i n.

• Gai nes Co

., TX : Observati on s w ere m ad e be tw

ee n pl an ti ng and harvest f ou r ti me s du ring t he

s

ea son

. Co ope rato r observed a d if fer en ce in the ea r ly

emer gen ce of the cotton, with the n on tr

an sgen ic li ne emerging at 4.88 plan ts/ft. ( 81 % ge rm ina ti on ) vs. 2.87 pl an ts/f t. (47 %

ge rm i na ti on ) fo r t

he t ra nsgen ic li ne s at the 1-2 l eaf stage of gro wt h. Observa ti ons taken on e

m

on th

late r s howed that this d iff e re n ce

was no lon ge r pres en t, with the tr an sg en ic li ne s havi ng

c

au ght up to the n on -tr an sg en ic li ne s. No ot he r d iff e ren ce s in d is ease, insect pe sts, or ph e no typ ic

a l d if fer en ces w er

e s ee n be tw

een the t ra nsg en ic and non -t ra nsgen ic li nes.

### B T

- 5A

- C o tt

o

n

- MR 2 05

- 0

4

0

- 0

6n • Uv al de Co., TX : Observa ti on s w ere ma de be tw

een plan ti ng and ha rvest f ou r ti mes du ring t he

s

ea son

. Co ope rato r observed differ en ces be tw

een t ran s

gen ic an d non-tr an sg en ic l i ne s with

tr

an sgen ic li ne s emerging at cotyle do n stage at 45% and the n on -tr an s

gen ic count er

pa rt at

7%

. Th is d iff e re nce wa s l ess no tic

e

ab le wi th in 14 days of the previ ou s ob serva ti ons, bu t still

ha d a st an d wh ich was 5-6% gr ea te r t han the non-trans gen ic li ne

. Bo th li ne s s ho wed e qual

infesta ti on s of Texas root ro t and Bo ll D a ng le, with an e qua l morta lity ra te of pl an ts

wh ich

c

on tracted ro ot rot

. A dd iti o na lly, tr an sgen ic li ne s s howed a mu ch

lowe r i nfesta ti on of cott on bo llw o

rm s, with infesta ti on rate s in the non-tr an s

gen ic l i ne s ranging fro m 20- 500 % gr ea t er infesta ti on than the t ra nsgen ic v arie ti es. No ot he r d iff er

en ces were noted in we ed iness

c

ha racter istics, ph e no ty

pe , o r be ne fici a l i nsect p opu lati on s.

• W ha rt on Co

., TX : Observati on s w ere ma de be tw

een pl an ti ng and harvest f ou r ti me s du ri ng
the s ea son

. Cooperator no t ic

ed little to no d if fer en ces be tw

een tr an sgen ic and n on -tr an sgen ic

li ne s, with a sli gh t de cli ne in on e tr an sfo rm a ti on event of th is t ra it (m ost lik e ly a tt ribut ab le

to t he pl an t varie ty

)

. Co ope rato r d id no ti ce that the tr an sg en ic pl an ts

ap pea red to be more of an

“ ope n plant ty pe” bu t th is cou ld not be assigned to e it he r t

he pl an t variety or the t ra nsgen ic

tra it. Bo llw or

m pressure ne ve r de vel op ed fu lly en ough to eval ua te the e ffic acy of the P IP tra it.

Pla nt Di s po sit io n : Pl an ts w ere harvested betw ee n la te Se ptem be r and lat e
Novem be r of 2 005 . A ll fi e ld sit e

s w ere de stroyed with no see d

retained

. Remaining plant materia l in the fi eld was cult iv

ated in to

the so il aft er ha rvest

. A ll s eed sent to co ope rator s w as u tiliz ed in the pl an ting of these tria ls.

V olun t ee r M oni t oring : Vo l un t ee r mo n it oring is cu rr en tly being con du cted on the Pi nal

Co

., Gai ne s Co

., and Uv alde Co. tria l si tes. Mo n it oring is

sc he duled to conclude one y ea r f ro

m t he dates of ha rve st.

M

on it oring was d isc on ti n ue d at the W ha rton Co. site on 5/19/0 6

du e to the re plant of anot he r r

egu lated ar ticle in the s am e tri al

ar

ea

.

W

ee din ess Ch aracteristics : T he re was no i nd ic a ti on of incr ea sed weedi ne ss c ha racter istics in

e it he r the tr an sg en ic or no n-tr an sgen ic v arie ti es.

Non -ta rg e t Org a ni sms : T he re was no i nd ic a ti on of an y adverse effe cts to non-tar get insect p opu la ti on s o r b en e

fici a l i nsects. Va ri ou s be ne ficia l i nsect

po pula ti on s w ere present with lace wi

ngs, assassin bug s, l ady

bee tles, and vari ou s c ol eo pteran species ob serve d.

W

ea ther Sy nop sis :

• Pi na l C o

., AZ : R epo rted drie r t han no rm a l f a ll s eas on

.

• Gai nes Co

., TX : Reported as no rm a l f o

r th is r eg ion of Texas.

### B T

- 5A

- C o tt

o

n

- MR 3 05

- 0

4

0

- 0

6n • Uv al de Co., TX : Ab no rm a lly dr y t he en ti re s ea s on

, with the mo n

th

of Ju ly be ing abno rm a lly ho t.

• W ha rt on Co

., TX : Ab no rm al ly ho t a ll s ea son, with dry c ond iti ons existing fro m J un e to mid-Ju ly.

Con ta inm e nt M eas ur es : A ll plots, with the exc ep ti on of the site in Gai ne s C o

., TX, wer e is

olated fro m ot he r s ex ua lly com pa ti b le

s

pe cies and c omm erci al

co tt

on by a d is t an ce of at least 660 f ee t. The Gai ne s Co. site was is

olated fro m t he su rr ou nd ing cotton by an is olation bo r de r of at

l ea st 40 f ee t. Th is is olation bo r de r w as va li da ted b y i nt er

na l aud it.

Si gna ture: Date: 6/30/0 6

Cy nth ia A. A ll en
R egu lator y C omp li an ce Specia list

Ba ye r C ropSci en ce – Re gu latory Affairs Bi oS ci en ce

### B T

- 5 D

-

C o tt

on

- MR 1 05

- 0

9

1

-

0

8

n US DA Fi e ld T e rmi nat ion R e port

US DA No tifi ca t ion Numb er : 05 -091-08n

Appli ca nt In te rn al Numb er : BT-5D-Co tt

on

- MR

Appli ca nt : Ba ye r C ropSci en ce LP

Res ea rch Triang le Park, N C 27 7 09 ( 919 ) 5 49 -2655

Re gul at ed Ar ticle : Gossy pu im hirsutum; plant incor po ra ted protectant (PIP); toleran t

to

glufosinate-a mm on ium herb ici de

Site Rel ea se In f orm at

io n : Tria ls u tilizi ng th is t ra it were c on du ct

ed at six (6 ) l ocations:

Coun ty / Sta te A c r ea ge
P l a n t ed D a t e
P l a n t ed D a t e
T e rmin a t ed Is ol at ion Met hod

C r itt

end e n C o

., AR 0

. 24 5 / 2

4

/ 05 11 / 1 /

05 66 0 ft. s pa ti a l is o

l a ti on
J a cks o

n C o

., A R 0

. 2

9

3 5 / 2

6

/ 05 11 / 4

/ 05 66 0 ft. s pa ti a l is o

l a ti on
Tift C o

., G A 0

. 29 6 / 2

1 /

05 12 / 1 4 / 0

5 66 0 ft. s pa ti a l is o

l a ti on
T a te C o

. , M S 0

. 17 5 / 3

0

/ 05 10 / 2

8

/ 0

5 66 0 ft. s pa ti a l is o

l a ti on
H o ckl e y C o

., T X 0

. 27 5 / 2

6

/ 05 11 / 2

1 /

0

5 66 0 ft. s pa ti a l is o

l a ti on
W

ha r t o

n C o

., TX 0

. 26 6 / 8

/ 05 11 / 1 4 / 0

5 66 0 ft. s pa ti a l is o

l a ti on

P urpo se of R el ease : Tria ls w ere estab lis

he d to eval ua te the perfo rm an ce of Pl ant

Incor po rated Protectant trait s in vari ou s cotton varie ti es, an d

ge nera te samples fo r residue analysis of co tt

on varieties.

Ob se rvat ion s :

• Cr itt

end en Co

., AR : C oo per ato r noted si gn ific ant reduc ti on in the ge rm i na tion rate of th e

tr

an sgen ic plo ts t h ro ug ho ut the trial pe ri od , a lt hough non-tr an sg en ic c he cks wit h in the pl ot

were a lso be low average germ i na ti on i nd ica ti ng po ssib le iss ue s with the eval ua ted varie ty. No

d iff er

en ce s w ere no ted in pl an t d is ea ses, insect pest po pu la ti ons, bene fici a l i nsect po pula ti ons,

o

r w eed ine ss c haracter isti cs. Coo pe rato r d id no te that tr an sgen ic v ariety in iti a lly s eemed to lag b eh ind the n on -tr an sg en ic v arie ty in vigor, but late r c au ght up, an d was gro wi

ng no rm a lly.

T he re was a lso a 15

- 25 % d iff er

en ce the bo ll op en ing be tw

ee n the tr an s

gen ic and n on tr

an sgen ic v arie ti es, wh ich the cooperato r a tt ri bu ted to the low pl an t po pu lation of th e

tr

an sgen ic v ariety.

### B T

- 5 D

-

C o tt

on

- MR 2 05

- 0

9

1

-

0

8

n • Jackson Co

., AR : Coo pe rato r no ted sign ific ant reduction in the ge rm i na ti on rate of t he

tr

an sgen ic plo ts t h ro ug ho ut the trial pe ri od , a lt hough non-tr an sg en ic c he cks wit h in the pl ot

were a lso be low average germ i na ti on i nd ica ti ng po ssib le iss ue s with the eval ua ted varie ty. No

si gn ific an t d iff er

en ces were no ted in pl an t d is eases, insect pest p opu la ti on s, b en e

fici a l i nsect

po pula ti ons, o r w e

ed iness c ha racteristics. Coo perato r d id no te

t

ha t tr an sgen ic plants

ap p

ea red to be s ho rter and d id not a pp ea r as hea lt hy as non -t ra nsgen ic plants, bu t m en ti on ed

th is c ou ld ha ve been due to the th in pl an t sta nd.

• Tift C o

., GA : C oo perato r no ted sign ific an t reduction in the ge rm i na ti on ra te of the tr an sg en ic plots thr ou ghout the trial pe ri od , altho ug h non-tr an sg en i c c he cks wit h in the plot were a lso

be lo w average ge rm i na ti on i nd ic a ti ng po ssib le iss ue s with the eval ua ted varie ty. No si gn ific a nt

d iff er

en ce s w ere no ted in pl an t d is ea ses o r bene fici a l i nsect p opu la ti on s. Coo pe rato r d id no te

si gn ific an t r

edu c

ti on s in b

ud wor m an d bo llw or

m po pulati on s in the tr an s

gen ic variety, and a lso

no ted a highe r perc en t ag e of plan ts with mu lti p le

te rm i na ls in the non -t ra nsg en ic. Th is t r end

was no ted in the n on -tr an sg en ic v arie ty

a s w e ll, but to a lesse r degr ee , i nd ic a ti ng thi s to be a varie ti a l effect, ra t he r than a tr an sg en ic o ne . Coo pe rato r a lso noted that t ra nsgen ic variety

s

ee med to ou t pe rfor m n on -tr an sgen ic v arie ty in gro wth an d fru it set.

• Ta te Co., MS : C oo perato r no ted sign ific an t r

edu ction in the ge rm i na ti on ra te of the tr an s

gen ic plots thr ou ghout the trial pe ri od , altho ug h non-tr an sg en i c c he cks wit h in the plot were a lso

be lo w average ge rm i na ti on i nd ic a ti ng po ssib le iss ue s with the eval ua ted varie ty. No si gn ific an t

d iff er

en ce s w ere no ted in pl an t d is ea ses, insect pest po pu la ti ons, bene fici a l i nsect po pula ti ons, ph ysica l plant c ha racteristics, o r w eedi ne ss c ha racteristics.

• Hockle y C o

., TX : C oope rato r no ted sign ific ant reduction in the ge rm i na ti on rate of t he

tr

an sgen ic plo ts t h ro ug ho ut the trial pe ri od , a lt hough non-tr an sg en ic c he cks wit h in the pl ot

were a lso be low average germ i na ti on i nd ica ti ng po ssib le iss ue s with the eval ua ted varie ty. No

si gn ific an t d iff er

en ces were no ted in pl an t d is eases, insect pest p opu la ti on s, b en e

fici a l i nsect

po pula ti ons, physica l plant c ha racteristics, o r w eedi ne s

s c ha racteristics.

• W ha rt on Co

., TX : Coo pe ra to r no ted sign ific ant reduction in the ge rm i na ti on rate of t he

tr

an sgen ic plo ts t h ro ug ho ut the trial pe ri od , a lt hough non-tr an sg en ic c he cks wit h in the pl ot

were a lso be low average germ i na ti on i nd ica ti ng po ssib le iss ue s with the eval ua ted varie ty. No

si gn ific an t d iff er

en ces were no ted in pl an t d is eases, insect pest p opu la ti on s, b en e

fici a l i nsect

po pula ti on s. Co ope rato r did no te

t

ha t tr an s

gen ic varieties had ver y l ow emergence, and d id

no t mature no rm a lly t h ro ug hou t the s ea s on . Th is resu lt ed in lo w reten ti on of fru it, incr ea se d

v

ege ta tive gro wt h, and s ho wed an incr ea sed inci den ce of hard lock.

Pla nt Di s po sit io n : Fo r a ll six l oca ti ons, plots were ha rvested and da ta taken, with sample s s ent to Ba ye r f a cili t y f o

r analysis. A ll remaini ng ha rvested materia l in the fi e ld and in the lab was de st

ro

y

ed.

U np l an ted s ee d wh ich was no t u tiliz ed by the co ope ra to r w as

e it he r returned to Ba ye r f or stor ag e o r destroye d.

### B T

- 5 D

-

C o tt

on

- MR 3 05

- 0

9

1

-

0

8

n V olun t ee r M oni t oring : Vo l un t ee r mo n it oring is o

ng oing fo r a ll t ria l sit es and is sc he dul ed

fo

r c omple ti on one y ea r f ro

m t he da te of ha rvest.

W

ee din ess Ch aracteristics : T he re was no i nd ic a ti on of incr ea sed weedi ne ss c ha racter istics in

e it he r the tr an sg en ic or no n-tr an sgen ic v arie ti es fo r mo st plo ts.

Mu lti p le

te rm i na l gro wth in bo th

varie ti e s in Tift Co

., GA was

no ted, al on g with the st un ted de vel op me nt of the t ra nsg en ic

varie ty in W ha rton Co

., TX. In bo th

instances, th is devel op m

en t

was a tt ributed to the plant variety.

Non -ta rg et Org a ni sms : T he re was no i nd ic a ti on of an y adverse effe cts to non-tar get insect p opu la ti on s o r b en e

fici a l i nsects. Va ri ou s be ne ficia l i nsect

po pula ti on s w ere present with lace wi

ngs, assassin bug s, l ady

bee tles, and vari ou s c ol eo pteran species ob serve d.

W

ea ther Sy nop sis :

• Cr itt

end en Co

., AR : Be lo w no rm a l rainfa ll; co mp en sated via i rr i ga ti on of plots.

• Jackson Co

., AR : No rm a l w ea the r c ond iti ons

• Tift C o

., GA : S li gh tly elevated rainfa ll am oun ts in late su mm er

• Ta te Co., MS : Be lo w average rainfa ll

• Hockle y C o

., TX : W ea t he r w as wit h in no rm al average s f o

r th is r eg i on ; a ha il st or

m w as

r epo rted ear ly in the season with minima l (<5% ) da mage to plots.

• W ha rt on Co

., TX : Plots ex pe ri en ced abo ve aver ag e te mp eratures fo r th is r eg i on . Mi no r plo t

da mage resulted fro m H u rr ic

an e Ri ta on 9/ 24 / 05

, bu t an eval ua ti o n

s

ho wed that t he re was no loss of li nt o r s eed fro m the plo t.

Con ta inm e nt M eas ur es : A ll plo ts w ere is olated from ot he r co tt

on by a d ist an ce of at least

66 0 ft. from the plot pe rimeter.

Si gna ture: Date: 6/30/0 6

Cy nth ia A. A ll en
R egu lator y C omp li an ce Specia list

Ba ye r C ropSci en ce – Re gu latory Affairs Bi oS ci en ce

### IR

- 5A

- GH

- MR 1 05

- 1 8 9

- 0

7n US DA Fi e ld T e rmi nat ion R e port

US DA No tifi ca t ion Numb er : 05 -189-07n

Appli ca nt In te rn al Numb er : IR

- 5A

- GH

- MR

Appli ca nt : Ba ye r C ropSci en ce LP

Res ea rch Triang le Park, N C 27 7 09 ( 919 ) 5 49 -26 55

Re gul at ed Ar ticle : Gossypiu m hirsutum; plant incor po ra ted protectant (PIP); toleran t

to

glufosinate-a mm on ium herb ici de

Site Rel ea se In f orm at

io n : Tria ls u tilizi ng th is t ra it were c on du ct

ed at one (1 ) l ocati on :

Coun ty / Sta te A c r ea ge
P l a n t ed D a t e

s
P l a n t ed D a t e (

s

)

T e rmin a t ed Is ol at ion Met hod (s)

Sab ana Gran de

, PR 1.0 15 11 /15/05 5/4/ 06 Is o

l a ti o n bo r de r & ph ysic a l b a rr i er

P urpo se of R el ease : Th is t ria l w as est ab lis

hed to evalua te the pe rfo rm an ce of plan t

incor po rated protectant trait s in cotton varie ti es.

Ob se rvat ion s : C oope ra to r did not no te an y d iff e re nces be tw

een tr an sg en ic a nd

non -t ra nsgen ic v arie ti es othe r t han effects of the P IP on bo llw or

m

po pula ti on s. No othe r d if fer en ces in be ne fici a l insect p opu la ti on s, pl an t d is ea ses, physica l plant characteristics, o r w eed iness

c

ha racter istics w ere r epo rte d.

Pla nt Di s po sit io n : C oope ra to r c on fi rm ed t ha t a ll s ee d sent b y Baye r and not used in

the c re

a ti on of the tria l w as returned

. The plot was ha rvest ed

5/4/ 06 . A ll ha rvested materia l w a s s ent to the Baye r f ac ility in

Lu b bo ck, Texas. Residual pl an t ma teria l w a s i ncor po rated in to

the tria l si t e.

V olun t ee r M oni t oring : Vo l un t ee r mo n it oring is cu rr en tly being con du cted an d is

sc he duled fo r c omple ti on one yea r f ro

m t he da te of ha rvest.

W

ee din ess Ch aracteristics : T he re was no i nd ic a ti on of incr ea sed weedi ne ss c ha racter istics in

e it he r the tr an sg en ic or no n-tr an sgen ic v arie ti es.

### IR

- 5A

- GH

- MR 2 05

- 1 8 9

- 0

7n Non -ta rg et Org a ni sms : T he re was no i nd ic a ti on of an y adverse effe cts to non-tar get insect p opu la ti on s o r b en e

fici a l i nsects.

W

ea ther Sy nop sis : C oope ra to r reported no rm al gro wi

ng c ond it i on s fo r t

h is r eg ion.

P lot D amage : No da ma ge to plo ts w as rep ort ed.

Con ta inm e nt M eas ur es : Plot was c on tained u tilizi ng a 40 ft is ola ti on bo rd

er

. As a

secon da ry m ea sure to prev en t ge ne fl o w,

a nyl on /mesh scr een

c

ag e was erected wh ich en closed the tr an sg en ic pl an ts

. T he

c

ag e was erected be fore fl owering and remained un til harvest.

Si gna ture: Date: 6/30/0 6

Cy nth ia A. A ll en
R egu lator y C omp li an ce Specia list

Ba ye r C ropSci en ce – Re gu latory Affairs Bi oS ci en cePage 1 o f 4 

### USDA Field Termination Report

USDA Notification Numbe r : 06

- 047

- 02n Applicant

Reference Number : IR

- 6A

- GH -M R Applican t:

Bayer CropS ci ence LP Research Triangle Park, NC 27709

Regulated Articl e : Gossypium hirsutum (cott o

n)

Site Release Information : Trials utilizing this trait were conducted in fourteen (14) l ocations.

County / State Acreage

Planted Date Planted Date Terminated Isolation M e

thod

Bossier/ LA 0.70 5/31/06 11/14/06 Isolation Border Rows

Madison/ LA 1.28 6/05/06 12/05/06 Isolation Border Rows

Coahoma/ MS 1.28 6/06/06 10/06/06 Isolation Border Rows

Oktibbeha/ MS 0.648 5/30/06 10/10/06 Isolation Border Rows Washington/ MS (2

- l oca t i o

ns) 

### 2.34 5/30/06 &

6/06/06 11/28/06 &

12/18/06 Isolation Border Rows

Durham & Halifax/

NC 0

- - Trials were n ot initiated

Dillon/ SC (2

- locations) 

### 3.88 6/02/06 &

5/30/06 12/08/06 &

12/11/06 Isolation Border Rows

Gaines/ TX 0.73 5/26/06 12/05/06 Isolation Border Rows Lubbock/ TX (1) 0.165 5/24/06 12/14/06 Isolation Border Rows Lubbock/ TX (2) 0

- - Trial was not initiated

Uvalde/ TX (2

- locations) 

### 1.12 5/25/06 10/30/06 &

10/03/06 Isolation Border Rows

Wharton/ TX (2

- l oca t i o

ns) 

### 1.2989 6/08/06 11/14/06 Isolation Border Rows

Observations : listing of characteristics between the transgenic and non

- transgenic:

1. Bossier Pa., LA
 Cooperator noted no unusual characteristics.
 Seedling disease was dis c

overed in both transgenic and non

- transgenic.
 No differences in plant disease, ins e

ct pest or beneficial insect populations.
 Non

- transgenic slightly taller than transgenic.
 Transgenic h ad more “hook

- bill e d” bolls.

2. Madison Pa., L A :  No unusual characteristics.
 No differences in emergence, maturity, fl owering, plant disease, insect pest population, weedine s

s or beneficial insect population s.

IR -6A-GH-MR 06-047-02nIR

- 6 A

- G H

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- 047

- 02n 

### 3. Coahoma Co ., MS :  Approximately 120 days after planting the cotton, drift from a nearby field the cooperator was spraying, caused the site to prematurely defoliate in t o a total loss of yield. Therefore, crop was not harvested and was destroyed with a

mechanical shredder.
 Prior to defoliation there were no signifi c ant differences between the transgenic and non

- transgenic. Observations were considered normal.

4. Oktibbeha Co., M S :  Plots were artificially inf e

sted with cotton boll worms.
 Non

- transgenic plots were grown without artificial infe s

tation of the cotton boll

worms.  No significant differences were noted in plant diseases, insect pest population,

beneficial in s

ect populations, weediness or physical plant characteristics.

5. Washington Co., MS :  Cooperator noted that there were no signif i cant differences in plant diseases, insect pest and benefici a l insect populations.
 Everything appeared normal between t he transgenic and non

- transgenic from

the first true -l eaf stage to maturity.

6. Washington Co., MS :  Cooperator noted that dur i ng maturity the non

- transgenic had a better fruit load than the transgenic.
 No significant differences were noted in plant diseases, insect pest populations, and beneficial insect populations.
 No significant differences in emergen c

e, squaring, blooming or maturity.
 A delay in ha r vesting due to an unusual c ool and wet autumn

7. Durham Co., NC:
 Study cancelled due to business reasons therefore, planting was ne v

er initiated.

8. Halifax Co., NC:
 Study was cancelled due to receiving seed too late.IR

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- 02n 

### 9. Dillon Co., SC (1 ) :  Cooperator did note that transgenic varie t ies had seve r e delayed maturity and parrot beak non

- opened bolls at maturity.
 Agronomic performance deemed as unacceptable. Efficacy data was biased based on poor crop performance for other physiologi c al aspects of the line

tested.  No significant differences were noted in plant diseases, insect pest populations or beneficial insect populations.

10. Dillon Co., SC (2 ):
 Cooperator noted delayed maturity, parrot beak underdeveloped and non

opening bolls.
 Agronomic performance deemed as unacceptable.
 Genetic Effects are unacceptable and many bolls ne v

er opened.
 Efficacy data was biased based on poor crop performance
 There were no significant differences in plant diseases, insect pest , and

beneficial in s

ect populations.

11. Gaines Co., T X :  Cooperator noted no significant differences in plant di s eases, insect pest, and

beneficial in s

ect populations.
 It was noted that all other visual observations were normal.
 There was some hail but it did not cause damage or loss to the plot.

12. Lubbock Co., TX (1) :  Cooperator noted no significant differences in plant di s eases, insect pest, and

beneficial in s

ect populations.
 Observations show no significant differences between the transgenic and non

transgenic plots.

13. Lubbock Co., TX (2) :  Study cancelled due to business reasons therefore, planting was ne v

er initiated

14. Uvalde Co., TX (1) :  No significant differences were noted in plant diseases, insect pest or beneficial populations.
 Observations show that the transgen i c plot had delayed maturity and the cotton bolls were unacceptably small.IR

- 6 A

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- 02n 

### 15. Uvalde Co., TX (2) :  No significant differences were noted in plant diseases, insect pest or beneficial populations
 It was noted by the cooperator that t he boll size for the transgenic was far superior to the non

- transgenic

16. Wharton Co., TX (1) & (2 ):
 Cooperator noted early boll opening for transgenic and non transgenic.
 The transgenic plot showed resistance to bollworm, budworm infestations.
 No significant differences were noted in plant diseases, insect pest or

beneficial in s

ect populations.
 No unusual growth patterns.

Plant Dispositio n: Plots were harvested and data taken, with samples sent to Bayer facility for analysis. All remaini n g harvested material in the field was destroyed. Unplanted seed which was not utilized by the cooperator was either returned to Bayer for storage, or destroyed.

Volunteer Monitoring : Volunteer monitoring is currently being c onducted on all trial sites, and scheduled to conclude one year from the termination date.

Weediness Characteristic s : There was no indication of increased weediness chara c teristics in either the transgenic or non

- transgenic plots.

Non

- target Organism s : There was no indication of any adverse effects to non

- target insect populations or beneficial in sects. Various bene f icial insect populations were present with lacewings, assassin bugs, lady bugs, and various coleopteran species observed.

Weather: Weather for all sites was ei t her normal or hot and dry. However, Washington County, Mississippi autumn season was unusually cool and wet causing a delay in harvesting and Wharton County, Texas (2), July and October was extremely wet.

Signature: Date: 9/19/07

Regina J. Hayes Regulatory Complian c

e Specialist Bayer CropS ci ence – Regulatory Affairs BioS ci enceIR

- 6D

- GH

- M R 1 of 3 06

- 068

- 03n 

### USDA Field Termination Report

USDA Notification Numbe r: 06

- 068

- 03n

Applicant Reference Number : IR

- 6D

- GH -MR

Applican t: Bayer CropS ci ence LP Research Triangle Park, NC 27709

Regulated Articl e: Gossypium hirsutum (cott o

n)

Site Release Information : Trials utilizing this trait were conducted in ten (10) locations.

County / State Acreage

Planted Date Planted Date

Terminated Isolation M e

thod

St. Landry/ LA 1.84 6/12/06 11/16/06 Isolation Border Rows

Tate/ MS (2 l ocations) 1.46 5/31/06 11/ 10/06 Isolation Border Rows Washington/ MS 1.49 6/08/06 12/02/06 Isolation Border Rows Hockley/ TX (2 locations) 1.92 5/30/06 & 6/06/06 12/18/06 Isolation Border Rows

Uvalde/ TX (2 locations) 2.85 5/30/06 & 6/13/06 11/10/06 Isolation Border Rows

Wharton/ TX (2 locations) 4.61 6/13/06 & 6/14/06 11/14/06 &

11/20/06 Isolation Border Rows

Observations : listing of characteristics between the transgenic and non

- transgenic:

1. St. Landry Co., L A :  No unusual characteristics.
 No difference in emergence, both plots had early flowering, boll set and boll opening with some boll rot.
 No differences in plant disease, i n sect pest or beneficial insect populations

2. Tate Co., M S :  No significant differences were noted in plant diseases, insect pest and

beneficial in s

ect populations.
 Cooperator did note that maturity of bolls were signi fi c antly slower in the transgenic plot as compared to the non

- transgenic.
 Seedling Vigor in the transgenic plot was approximately 40% of the non

transgenic.IR

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- GH

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- 03n 

### 3. Washington Co., MS :  Cooperator stated that over

- all the plots are similar and there are no signifi c ant differences.
 The plots yield was poor and the transgenic appears to have slightly less fruit

set.  Misshaped bolls are present in transgenic and non

- transgenic plots
 T he hot dry weather seemed to stress t h

e plants and as a result fruit shedding

occurred.
 Regimen A has set more fruit than Regimen B or C.
 Regimens B and C had poor yields.
 No significant differences were noted in plant diseases, insect pest, and

beneficial in s

ect populations.

4. Hockle y Co., T X :  Cooperator stated there were no diff e rence in emergence and no significant differences in plant disea s es, ins e

ct pest populations, beneficial insect populations, emergence, flower i ng, boll s et and boll opening.

5. Uvalde Co., TX :  No significant differences were noted in plant diseases, insect pest, beneficial insect and wild life populations.
 Due to low yield (15%) emergence) a decision was made to re

- plant. Replanting took place two (2) weeks lat e r. During the re

- planting, previou s

ly emerged plants were not removed.  It was noted that the transgenic was less tolerant of whiteflies than the non

transgenic. Possible, two

- week diffe r ence in planting date may have been the

cause.  It w as observed that the transgenic cotton was very light and fluffy, there was no worm presence but the whiteflies m a de up for the l ack of worm presence.

6. Wharton Co., TX :  Cooperator noted no unusual characteristics/growth patterns.  No significant di fferences were noted in plant diseases, insect pest or

beneficial in s

ect populations.
 Early bloom for transgenic and non

- transgenic.
 Transgenic emergence was lower than expected and the non

- transgenic had good emergence.
 Bolls in transgenic were s maller than normal

Plant Dispositio n : Plots were harvested and data taken, with samples sent to Bayer facilities for analyses. All remaini n g harvested material in the field was destroyed. Unplanted seed which was not utilized by the cooperator was either returned to Bayer for storage, or destroyed.IR

- 6D

- GH

- M R 3 of 3 06

- 068

- 03n 

### Volunteer Monitoring : Volunteer monitoring is currently being c onducted on all trial sites, and scheduled to conclude one year f r om the termination date.

Weediness Characteristic s: There was no indication of increased weediness chara c teristics in either the transgenic or non

- transgenic plots.

Non

- target Organism s: There was no indication of any adverse effects to non

- target insect populations or beneficial in sects. Various bene f icial insect populations were present with lacewings, assassin bugs, lady
beetles, and various coleopteran species observed.

Weather : Weather for all sites were either normal or hot and dry. Wharton County, Texas experienced an extremely wet season for the months of July and October.

Signature: Date: 9/19/07

Regina J. Hayes Regulatory Complian c

e Specialist Bayer CropS ci ence – Regulatory Affairs BioS ci enceAppendix 2. Materials and Methods2.A

. Insert Characterization

Southern analyses were conducted to characterize the DNA insertion in event T303

- 3 cotton. Genomic DNA was isolated from transgenic and non

- transgenic plants and these samples were subjected to Southern analyses using the different components of the transgenic cassette (3’me1, cry1Ab , P35S

, bar , 3’nos, 5’e1) as well as the complete T

DNA fragment as probes.

Test Items, Reference Items, and Standards

Test Item

BC 2 F 2 of FM966 event T303

- 3 was grown in the greenhouse and leaf tissues were harvested from individual plants for genomic DNA extraction.

Control Cotton Material

Non

- transgenic cotton variety FM966 seed were planted in the greenhouse and leaf tissues were harvested from individual plants for genomic DNA extraction.

Reference Material Plasmid DNA from pTDL004 was prepared from E.coli strain MC1061.

λ DNA digeste d with Pst I (Fermentas, Burlington, Ontario, Canada) was used as the molecular weight marker on the agarose gels.

Genomic DNA Extraction

Total genomic DNA was prepared from leaf material harvested from event T303

- 3 cotton

and from leaf material of non

- transgenic cotton variety FM966. The frozen tissue samples were pulverized and the DNA was extracted using milli

- Q (MQ) water and

chloroform :isoamylalcohol. To remove all undissolved particles present in the DNA samples, the final DNA samples were centrifuged. All samples had large, viscous pellets. The supernatant was removed to a clean tube and the pellets were dissolved in MQ

water overn ight. The concentration of the DNA samples was determined.

Restriction Digests of Total Genomic DNA

For each Southern blot, nine aliquots of 10 μ g of event T303

- 3 genomic DNA were digested with the following restriction enzymes: Bam HI, Bgl II, Sac I, Kp n I, Nco I, Pvu I,

Xba I, Eco RV and Not I. Two aliquots of 10 μ g genomic DNA from the non

- transgenic

control were digested with the Not I restriction enzyme. The total reaction volume was 50

μ l and the digests were incubated overnight at 37 o C.

Five μ g of plasmid pTDL004 DNA were digested with Not I. The total reaction volume

was 50 μ l and the incubation was performed for approximately three hours at 37 ° C. Plasmid DNA digestion was checked by agarose gel electrophoresis and the four expected fragments (616 7bp, 4915bp, 1532bp and 1290bp) were observed. The concentration of the digested plasmid DNA was determined to be 95.33 ng/ μ l.Gel Electrophoresis of Digested DNA Samples

Four identical agarose gels were prepared. An appropriate dilution of the Not I digested pTDL004 sample was made. Assuming a single copy integration of the transgene into the cotton genome, 10 μ g of homozygous genomic DNA corresponds to ~ 30.9 pg of pTDL004 plasmid DNA. The amount representing approximately one plasmid copy per genome was added to 10 μ g of digested non

- transgenic DNA. This served as the positive control and was used to show that the hybridizations were performed under conditions allowing hybridization of the probe s with the target sequences.

Af ter addition of the loading dye, each digest was loaded onto the gel. λ DNA digested

with Pst I was included as the molecular weight standard. Following electrophoresis, the separated DNA fragments were transferred from the agarose gel to a Hybond

- N+

memb rane.

DNA Probe Hybridization

Probe templates each containing one of the genetic elements of plasmid pTDL004 and the complete T

- DNA probe were prepared by PCR. Identical PCR reactions were pooled and following volume reduction by evaporation, the different PCR products were loaded on an agarose gel and purified by gel extraction. To determine the qualit y of the probe templates, samples of each were loaded on an agarose gel and electrophoresed. The determined molecular weight of each probe template fit with the expected fragment sizes. The concentration of the different probe templates was determined by comparing band intensities of the purified probe templates with band intensities of a low DNA mass ladder (Invitrogen Corp., Carlsbad, CA). The templates were stored in the freezer.

For hybridization, the probe templates were labeled with [ α

32 P]. Visualization of the hybridizing fragments was achieved via exposure of a BioMax MR film to the hybridized

membrane.

Stripping of Probes

Membranes were stripped of the radioactively

- labeled probes to prepare the blot for subsequent re

- hybridization to additional probes.

Determination of Hybridization Fragment Sizes

Lengths of all hybridization fragments were determined by comparing them with the molecular weight marker. A semi

- logarithmic standard curve was constructed using the λ DNA marker with the y

- axis being the distance between the gel loading slot and the different fragments of digested marker DNA and the sizes of the fragments of the DNA

marker on the x

- axis. The length of each hybridization fragment was determined by measuring the migration distance of the fragment and extrapolating it to the standard

curve.

2.B. Confirmation of Absence of Vector Backbone

Southern analyses were conducted to confirm the absence of vector backbone from plasmid pTDL004. Genomic DNA was isolated from event T303 -3 plant material andprobed with overlapping elements of the vector backbone. Samples were then probed with fragments of the T

- DNA ( p35S and cry1Ab ) as a positive control.

Test Items, Reference Items, and Standards

Test Item

BC 2 F 3 of FM966 event T303

- 3 was grown in the greenhouse and leaf tissues were harvested from individual plants for genomic DNA extraction.

Control Cotton Material

Non

- transgenic cotton variety FM966 seed were planted in the greenhouse and leaf tissues were harvested from individual plants for genomic DNA extraction.

Reference Material Plasmid DNA from pTDL004 was prepared from E.coli strain MC1061.

λ DNA digested with Pst I (Fermentas, Burlington, Ontario, Canada) was used as the molecular weight marker on the agarose gels.

Genomic DNA Extraction

Total genomic DNA was prepared from leaf material harvested from event T303

- 3 cotton

and from leaf material of non

- transgenic cotton variety FM966

.

Restriction Digests of Total Genomic DNA

Seven samples each of 10 μg of T303

- 3 genomic DNA were digested with Not I or Eco RV restriction enzyme s and 28 samples each of 10 μg non

- transgenic control genomic DNA were digested with the Not I restriction enzyme. All digestions were performed in a total reaction volume of 40 μl. The digests were incubated overnight at 37°C.

Five μg of pTDL004 plasmid DNA was digested with NotI. The total reaction volume was 50 μl. Incubation was performed overnight at 37°C. To ch eck that the plasmid DNA was totally digested, 200 ng of the digest were loaded on a 1% TAE horizontal submarine agarose gel. All fragments separated as expected (6167 bp, 4915 bp, 1532 bp and 1290 bp). The concentration of the Not I

– digest ed plasmid DNA was determined at 98.56 ng/μl by measuring the fluorescence of the Quant

- iTTM PicoGreen® dsDNA reagent.

Gel Electrophoresis of Digested DNA Samples

Seven identical agarose gels were prepared. An appropriate dilution of the Not I digested plasmid pTDL004 sample was made. Assuming a single copy integration of the transgene into the cotton genome, 10 μg of homozygous genomic DNA corresponds to ~ 30.9 pg of pTDL004 plasmid DNA. The amount representing approximately 0.1, 1 and 10 plasmid copies per g enome was added to 10 μg of digested non

- transgenic DNA. These preparations served as the positive control s and were used to show that the hybridizations were performed under conditions allowing hybridization of the probe s with the target sequences.

Each digest was loaded onto the gel in its appropriate well. λ DNA digested with Pst I was included as the molecular weight standard.Following electrophoresis, the separated DNA fragments were transferred from the agarose gel to a Hybond

- N+ membrane.

DNA Probe Hybridization with Vector Backbone Probe Templates

The vector backbone probes were prepared by [α

32 P] labeling of probe templates PT002, PT003, PT005, PT007, PT008, PT009 and PT01 0. Each of the seven membranes was hybridized with a different [α

32 P] labeled probe

. Visualization of the hybridizing fragments

was achieved via exposure of a BioMax MR film to the hybridized membrane.

Following the stripping of vector backbone probes from the membranes, each membrane

was re

- hybridized with the T

- DNA probe . The T

- DNA probe was prepared by [ α- 32 P] labeling. Visualization of the hybridizing fragments was also achieved via exposure of a BioMax MR film to the hybridized membrane.

Determination of Hybridization Fragment Sizes

Lengths of all hybridization fragments were determined by comparing them with the molecular weight marker. A semi

- logarithmic standard curve was constructed using the λ DNA marker with the y

- axis being the distance between the gel loading slot and the

dif ferent fragments of digested marker DNA and the sizes of the fragments of the DNA

marker on the x

- axis. The length of each hybridization fragment is determined by measuring the migration distance of the fragment and extrapolating it to the standard

curve.

2.C

. Protein (Cry1Ab and PAT) Expression in Raw Agricultural Commodity

Fuzzy seed (ginned seed but not yet de

- linted) from event T303 -3 cotton were analyzed

for the expression of Cry1Ab and PAT proteins using enzyme

- linked immunosorbent assays (ELISAs).

Field production of Seed Samples

Generation T 4 e vent T303

- 3 cotton seed and non

- transgenic Coker 315 cotton seed were grown in six trials in the U.S . All trials were managed according to regional commercial production practices

. S amples of fuzzy seed were collected from ginned harvested samples from each plot at each trial site.

Reference Materials

Cry1Ab and PAT proteins, produced in E.coli , were used as standards for the ELISA

methods.

Processing of Fuzzy Seed Samples

The fuzzy seed was processed by mechanical de

- linting. The de

- linted seed was mechanically cracked in a roller mill and the hull material was separated from the kernel.Protein Extraction from Samples

The sample materials were prepared by either grinding in a blender pre

- chilled with dry ice or by grinding with a mortar and pestle. The ground sample was mixed with

extraction buffer and shaken for 30 min at 4 ° C. Insoluble material was removed by centrifugation. The supernatants were then analyzed by the specific ELISAs for the presence of the Cry1Ab and PAT proteins.

Determination of Cry1Ab and PAT Protein Concentrations

The amount of Cry1Ab and PAT protein in the sample extracts were determined by commercial ELISA kits (EnviroLogix, Inc., Portland, ME). All assays resulted in color development in the last step, which was measured by optical density of each microtiterplate well at 450nm. Softmax Pro TM software (Molecular Devices, Vers i on 4.0)

was used to derive the concentration of immunoreactive Cry1Ab and PAT protein s . The optical density (OD) values were adjusted for the buffer blank and then any background due to the matrix was subtracted, using the average value from two wells containing non

transgenic extracts, assayed on the same microtiter plate. The OD corrected for the

buffer blank and the non

- transgenic background was converted to the protein analyte concentration using the standard curve from the same microtiter plate.

2.D. Protein Characterization

Because a full equ ivalence study ( USDA, 2008) was done on the Cry1Ab protein isolated

from TwinLink C otton (where event T304

- 40, the antecedent organism, is one of the parent s in the TwinLink breeding stack), only an SDS

- PAGE analysis was done in this study to compare Cry1Ab protein produced in event T303

- 3 to that produced in E. coli

and event T304

- 40.

Test Items, Reference Items, and Standards

Protein expressed in planta

E vent T303

- 3 and event T304

- 40 plants were grown in the greenhouse and leaves were harvested into plastic bags and placed on dry ice. Leaf samples were processed in a Waring blender pre

- chilled with dry ice, with small amounts of dry ice added during the blending process to ensure samples remained frozen. Ground samples were stored in a freezer at approximately

- 20 °C overnight to allow for dry ice dissipation prior to extraction of protein.

The Cry1Ab proteins were extracted by mixing ground plant leaves with extraction buffer

at a ratio of 25 g of ground leaves to 100 mL of extraction buffer in 5 oz specimen

containers. The 850 mL of extraction buffer contained 150 mM NaCl and 100 mM sodium phosphate (pH 7.2), 0.85 g of polyvinylpyrrolidinone (40,000 Mol. Wt), 1 mM PMSF, 1

mM benzamidine/caproic acid and 850 μg of leupept in/antipain.

After addition of the extraction buffer to the ground leaf material, the 5 oz container with extraction buffer and ground leaf material was continuously inverted on a rotary rocker at ~30 rpm for 20 minutes at room temperature. The extract wa s filtered through Miracloth® and the filtrate vacuum filtered through a 0.45 μ m cellulose acetate membrane.The Cry1Ab antibody affinity columns used for the purification were prepared using approximately 750 μL of crude rabbit polyclonal antibody serum raised against the Cry1Ab protein. The Cry1Ab antibodies were covalently attached to individual Pierce

AminoLink® Plus immobilization columns

.

Protein Expressed by E. coli

T rypsin

- digested Cry1Ab protein (batch no. 25.05.99.70, purity > 85% ) expressed by E.

coli was provided by Bayer BioScience N.V. Molecular and Biochemical Analytical

Services
- Protein Characterization, Zwijnaarde, Belgium

.

T hrombin

- engineered Cry1Ab protein from E.coli (batch no. Min1418

- 1, purity > 85%) was provided by Bayer BioScience N.V. Molecular and Biochemical Analytical Services
Protein Characterization, Zwijnaarde, Belgium.

Molecular Weight Markers

Molecular weight markers (Invitrogen BenchMark, Cat# 10747) comprised of a series of

recom binant proteins of known molecular weight.

SDS

- PAGE

The Cry1Ab protein from E. coli and the Cry1Ab protein isolated from events T303

- 3 and

T304

- 40 cotton leaves were analyzed by SDS

- PAGE. The protein from the plants and the corresponding protein from E. coli were denatured and analyzed by electrophoresis on a polyacrylamide gel

. The gel was stained with Coomassie Brilliant Blue R

- 250 to visualize the protein bands.

SDS

- PAGE analysis was also conducted as described above on trypsin

- digested Cry1Ab protein from E. coli

, the thrombin

- engineered Cry1Ab protein from E. coli , and Cry1Ab protein isolated from the leaves of event T304

- 40.

The electrophoretic mobility on a polyacrylamide gel of recombinant proteins of known molecular weight (Molecular Weight Markers) was utilized to generate a standard curve of electrophoretic mobility for each of the SDS

- PAGE analys es run s . The standard

curve s were utilized to determine the molecular weights of the Cry1Ab proteins from E.

coli (trypsin

- dig ested and thrombin

- engineered), event T304

- 40, and event T303

- 3.

2 .E. Composition Analysis

Samples were analyzed to assess the nutrient and anti

- nutrient composition of event

T 303 -3 cotton grain and compare it to that of the non

- transgenic control.

Field production of Seed Samples

Generation T 4 event T303 -3 and non

- transgenic cotton seed ( Coker 315 ) were grown in

six trials in primary cotton growing regions of the U.S in 2005. All trials were managed according to regional commercial production prac t ices. Each trial consisted of nine plots

.Six plots were event T303

- 3 and three were non

- transgenic Coker 315. Of the six event

T303

- 3 plots, three were treated twice with a tank mix of glufosinate ammonium herbicide and ammonium sulfate at 0.52 lb ai/A (equivalent to 40 oz Ignite ® Herbicide/A) and 3.0

lb/A , respectively. S amples of fuzzy seed were collected from each plot (i.e., three samples T303

- 3 sprayed, three samples T303

- 3 nonsprayed , three samples non

transgenic Coker 315) at each trial site an d shipped frozen to Bayer CropScience, RTP,

NC.

Composition Analysis of Raw Agricultural Commodity

Subsamples of frozen ginned seed were sent to two 3 rd party labs for composition analysis, Eurofins Scientific (Des Moines, IA) and Covance Laboratories (M adison, WI). All parameters with the exception of cyclopropenoid fatty acid content were performed by

Eurofins Scientific.

Methods utilized by laboratories for each parameter analyzed are shown in T able 2 1.

Statistical Methods

Analysis was conducted by analyzing for differences between the three treatments (i.e.,

event T303

- 3 sprayed, event T303

- 3 unsprayed, non

- transgenic Coker 315) for 55 different components. These components were:

• Proximate and fiber compounds : moisture, protein, crude fat, ash, total carbohydrates, ADF, and NDF

• Minerals : Calcium

, Phosphorus, P otassium , M ag nesium , Iron , and Z inc

• Tocopherols : alpha

- , beta -, gamma

- , and delta tocopherol, total tocopherol (V itamin E )

• Anti

- nutrients : free gossypol, “

- “ and “+” gossypol, total gossypol, phytic acid

• A mino acids : alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine

• F atty acids : myristic (C14:0), palmitic (C16:0), stearic C18:0), arachidic (C20:0), behenic (C22:0), lignoceric (C24:0), palmitoleic (C16:1), oleic (C18:1), linoleic (C18:2), linolenic (C18:3)

• Cyclopropenoid fatty acids : malvic acid, sterculic acid and dihydrosterculic acid

Statistical analyses included descriptive statistics (i.e., mean value, standard deviation, min/max values), over all analysis (i.e., ANOVA by treatment, site, and respective interaction terms), and by site analysis (i.e., ANOVA by treatment where signific ant interactions [p<0.05] by site were detected, followed by t

- tests). Level of significance was

fixed 0.05 (two

- sided).Table 21

. Analytical m ethod s u sed for compositional a nalysis

Parameter Laboratory Method

Moisture Eurofins Scientific AOAC 930.15

Crude fat Eurofins Scientific AOAC 920.39 Crude protein Eurofins Scientific AOCS 990.03

Ash Eurofins Scientific AOAC 942.05 Acid Detergent Fiber Eurofins Scientific ANKOM ADF 05/03 Neutral Detergent Fiber Eurofins Scientific ANKOM NDF 05/03

Carbohydrates Eurofins Scientific 21CFR

- Difference

between 100 and the sum of crude protein, fat,

moisture and ash

Ca Eurofins Scientific AOAC 984.27 and 985.01

P Eurofins Scientific AOAC 984.27 and 985.01

K Eurofins Scientific AOAC 984.27 and 985.01

Fe Eurofins Scientific AOAC 984.27 and 985.01 Mg Eurofins Scientific AOAC 984.27 and 985.01

Zn Eurofins Scientific AOAC 984.27 and 985.01

Tocopherols Eurofins Scientific AOAC 971.30 with HPLC
Quantitation

Amino Acids Eurofins Scientific AOAC 982.30 Sec. D, F

Fatty Acids Eurofins Scientific AOCS Ce 1e

- 91 (Capillary GC)

Phytic Acid Eurofins Scientific Analytical Biochemistry 77,

536

- 539 (1977)

Gossypol (total) Eurofins Scientific AOCS Ba 8

- 78 (1983, reapproved 1997)

Gossypol (free) Eurofins Scientific Method version of AOCS Ba

7- 58 (reapproved 1997) and JAOCS vol. 59, no. 12, pp.

546

- 549 (Dec. 1982) (Modified)

Cyclopropenoid Fatty Acids Covance Laboratories Covance method procedure

MP

- CPFA

- MA (HPLC)Appendix 3. Characterization of Event T303 -33.A. Insert Characterization

Genomic DNA isolated from event T303

- 3 cotton was digested with one of the following restriction enzymes: Bam HI, Bgl II, Sac I, Kpn I, Nco I, Pvu I, Xba I, Eco RV, and Not I. Non

transgenic genomic DNA digested with Not I was used as a negative control; non

transgenic genomic DNA supplemented with one copy of pTDL004 and digested with

Not I was used as a positive control. The resulting DNA fragments were analyzed by Southern blot with six different probes, corresponding to the different genetic elements

contained in the T- DNA from plasmid pTDL004. Pro be information is presented in T able

22

. An overview of the obtained Southern blot results is present in F igure 11

.

The results of the Southern blot analysis demonstrate that the inserted transgenic sequence in event T303

- 3 consists of one complete copy of the cry1Ab and bar gene s with the associated regulatory sequences required for expression.

Table 22

. Event T303

- 3 insert verification – probe i nformation Probe Template ID Genetic Element Size Probe Template (bp)

PT020

- 1 3’me1 probe 859

PT021

- 1 c ry1Ab probe 1822

PT022

- 1 P35S3 probe 801

PT023

- 1 bar probe 425

PT024

- 1 3’nos probe 214

PT025

- 1 Complete T

- DNA probe 4929Figure 11. Event T303

- 3 insert verification – Southern blot r esults

3’me1 probe **cry1Ab** probe

P35S3 probe **bar** probe

Loading Sequence

- All Probes

Lane 2: G. hirsutum event T303

- 3
– Bam HI digested

Lane 3: G. hirsutum event T303

- 3
– Bgl II digested

Lane 4: G. hirsutum event T303

- 3
– Sac I digested

Lane 5: G. hirsutum event T303

- 3
– Kpn I digested

Lane 6: G. hirsutum event T303

- 3
– Nco I digested

Lane 7: G. hirsutum event T303

- 3
– Pvu I digested

Lane 8: G. hirsutum event T303

- 3
– Xba I digested

Lane 9: G. hirsutum event T303

- 3
– Eco RV digested

Lane 10: G. hirsutum event T303

- 3
– Not I digested

Lane 11: G. hirsutum wild type
– Not I digested

Lane 12: G. hirsutum w ild type + 1 copy pTDL004

– Not I digestedFigure 11 (continued). Event T303

- 3 insert v erific ation – Southern blot r esults

3’nos probe

Complete T

- DNA probe Loading Sequence

- All Probes

Lane 2: G. hirsutum event T303

- 3
– Bam HI digested

Lane 3: G. hirsutum event T303

- 3
– Bgl II digested

Lane 4: G. hirsutum event T303

- 3
– Sac I digested

Lane 5: G. hirsutum event T303

- 3
– Kpn I digested

Lane 6: G. hirsutum event T303

- 3
– Nco I digested

Lane 7: G. hirsutum event T303

- 3
– Pvu I digested

Lane 8: G. hirsutum event T303

- 3
– Xba I digested

Lane 9: G. hirsutum event T303

- 3
– Eco RV digested

Lane 10: G. hirsutum event T303

- 3
– Not I digested

Lane 11: G. hirsutum wild type
– Not I digested

Lane 12: G. hirsutum wild type + 1 copy pTDL004

– Not I digested3.B. Confirmation of Absence of Vector Backbone

Genomic DNA isolated from event T303

- 3 cotton was digested with restriction enzymes

Eco RV and Not I. Non

- transgenic genomic DNA digested with Not I was used as a negative control; non

- transgenic genomic DNA supplemented with 0.1, 1, and 10 copies

of plasmid pTDL004 and digested with Not I was used as a positive control. The resulting DNA fragments were analyzed by Southern blot with seven overlapping probes, corresponding to the different genetic elements contained in the vector backbone of plasmid pTDL004 as well as the complete T

- DNA probe. Probe in f ormation is presented

in Table 23

.

Table 24 and 25 summarizes the expected and obtained hybridization fragments using vector backbone probes and T

- DNA probe, respectively

.

The results of the Southern blot analysis confirm than event T303

- 3 contains n o elements of the pTDL004 vector backbone.

Table 23. Event T303

- 3 vector backbone analysis – probe i nformation

Probe Template ID Genetic Element Position in pTDL004 Size Probe Template (bp)

PT007 -1 Vector Backbone

Probe bp 5135 to 6480 1346

PT002 -1 Vector Backbone

Probe bp 5981 to 8019 2039

PT003 -1 Vector Backbone

Probe bp 7474 to 9461 1988

PT008 -1 Vector Backbone

Probe bp 8794 to 10860 2067

PT009 -1 Vector Backbone

Probe bp 10277 to 11629 1353

PT010 -1 Vector Backbone

Probe bp 10931 to 12526 1596

PT005 -1 Vector Backbone

Probe bp 12019 to 13879 1861

PT011 -1 T

- DNA probe (P35S + cry1Ab) bp 1619 to 4187 2492Table 24. Expected and obtained hybridization fragments with overlapping vector backbone probe t emplates

Fragment sizes are provided in bp *Only 85 bp overlapTable 25. Expected and obtained h ybridizat ion fragments using T

- DNA p robe

Fragment sizes are provided in bp3.C. Composition Analysis

Fuzzy seed was harvested from six field trials

. Each trial consisted of three replications

of event T303

- 3 not sprayed , event T303

- 3 sprayed and the non

- transgenic Coker 315. Fuzzy seed samples were processed and analyzed for proximate, amino acid, fatty acid, cyclopropenoid fatty acid, mineral, tocopherol, and anti

- nutrient composition. Data for

comp osition analysis is present ed in T able 26 , 27, 28 and 29.

Table 26

. A mino acid composition in cottonseed of event T303 -3

To tal A min o Aci ds % Dry m atter

Refere nc e

ranges a N o n

Transg e n ic Transg e n ic

Not sp ra y ed Transg e n ic Sprayed

Mean ± SD Mean ± SD Mean ± SD

Alanine 0

. 9 4 ± 0

. 08 0

. 9 9 ± 0

. 07 1

. 0 0 ± 0

. 06 0

. 4 2

- 1

. 5 1

A r gin i ne 2

. 4 8 ± 0

. 33 2

. 6 2 ± 0

. 28 2

. 6 5 ± 0

. 22 1

. 0 5

- 4

. 4 0
Asp a rtic acid 2

. 2 6 ± 0

. 21 2

. 3 0 ± 0

. 21 2

. 3 3 ± 0

. 17 1

. 0 0

- 3

. 5 5 Cysti n e 0

. 3 4 ± 0

. 04 0

. 3 6 ± 0

. 03 0

. 3 7 ± 0

. 03 0

. 1 6

- 0

. 8 6

Gluta m ic acid 4

. 6 4 ± 0

. 55 4

. 9 1 ± 0

. 47 4

. 9 7 ± 0

. 38 1

. 9 6

- 8

. 1 6

Gl y cine 0

. 9 5 ± 0

. 09 0

. 9 9 ± 0

. 08 1

. 0 0 ± 0

. 06 0

. 4 4

- 1

. 5 8

Histi d i n e 0

. 6 3 ± 0

. 07 0

. 6 7 ± 0

. 06 0

. 6 7 ± 0

. 05 0

. 3 1

- 1

. 0 3

Isol e u cine 0

. 7 0 ± 0

. 06 0

. 7 3 ± 0

. 07 0

. 7 5 ± 0

. 05 0

. 3 5

- 1

. 1 7

Leucine 1

. 3 3 ± 0

. 13 1

. 4 0 ± 0

. 12 1

. 4 2 ± 0

. 10 0

. 6 3

- 2

. 2 3 Lysine 1

. 1 1 ± 0

. 16 1

. 2 3 ± 0

. 15 1

. 1 8 ± 0

. 10 0

. 5 2

- 1

. 6 5

M et h ion i ne 0

. 3 8 ± 0

. 04 0

. 4 1 ± 0

. 03 0

. 4 1 ± 0

. 03 0

. 1 5

- 0

. 5 4

Ph e n ylalanine 1

. 2 2 ± 0

. 14 1

. 2 8 ± 0

. 13 1

. 3 0 ± 0

. 10 0

. 5 4

- 2

. 0 3

Prol i n e 0

. 9 1 ± 0

. 11 0

. 9 5 ± 0

. 09 0

. 9 7 ± 0

. 11 0

. 4 1

- 1

. 3 9

Serine 1

. 0 2 ± 0

. 10 1

. 0 8 ± 0

. 09 1

. 0 9 ± 0

. 07 0

. 5 0

- 1

. 6 3

Thr e onine 0

. 7 5 ± 0

. 06 0

. 7 9 ± 0

. 06 0

. 8 0 ± 0

. 04 0

. 3 4

- 1

. 2 1
Try p top h an 0

. 2 8 ± 0

. 03 0

. 2 9 ± 0

. 02 0

. 3 0 ± 0

. 02 0

. 1 0

- 0

. 4 9 Tyros i ne 0

. 5 6 ± 0

. 06 0

. 5 9 ± 0

. 06 0

. 6 1 ± 0

. 05 0

. 3 2

- 1

. 1 7

Vali n e 0

. 9 7 ± 0

. 10 1

. 0 2 ± 0

. 10 1

. 0 3 ± 0

. 08 0

. 4 5

- 1

. 6 7

Data re pr e sent an av er age of three r ep licate samp les at six field test sites.

a Refer e nce ra ng es co mp iled fr om OECD (2004), ILSI (2007), La wh on et al

. (1976), and Bertrand et al

. (2005)Table 27 . F atty acid composition in cottonseed of event T303 -3

To tal fatty acids % Relati v e

NonTransg eni Transg en ic

Not sp ra y ed Transg en ic Sprayed Refere nce ran g es a

Mean ± SD Mean ± SD Mean ± SD

**S**

**a tur**  **a ted**  My ristic C 14 : 0 0

. 7 0 ± 0

. 12 0

. 6 2 ± 0

. 07 0

. 6 1 ± 0

. 06 0

. 5 3

- 1

. 1 7

Pa l m i t ic C 16 :0 2 3

. 2 1 ± 1

. 38 2 2

. 4 3 ± 1

. 05 2 2

. 3 4 ± 1

. 07 21.1

- 29

. 9

StearicC18 : 0 2

. 4 2 ± 0

. 11 2

. 5 2 ± 0

. 12 2

. 5 1 ± 0

. 13 2

. 1 5

- 3 .4

Ara c hidic C 20 : 0 0

. 3 0 ± 0

. 02 0

. 2 8 ± 0

. 03 0

. 2 8 ± 0

. 02 0

- 0

. 48

Behenic C 22 : 0 0

. 1 7 ± 0

. 05 0

. 1 5 ± 0

. 01 0

. 1 5 ± 0

. 01 0

- 0

. 27 Lig n oceric C 2 4:0 0

. 1 2 ± 0

. 02 0

. 1 1 ± 0

. 01 0

. 1 1 ± 0

. 01 0

- 0

. 30

Sum sat u rated 2 6

. 9 2 2 6

. 1 1 2 6

. 0 0 2 4 .1

- 3 5

. 5

**Mono**

**- u**  **nsat ur**  **ated**

Pa l m it o leic C 16 :1 0

. 5 1 ± 0

. 04 0

. 4 8 ± 0

. 04 0

. 4 8 ± 0

. 04 0

. 4 6

- 0

. 8 6

Oleic C18:1 1 4

. 8 0 ± 0

. 77 1 4

. 6 1 ± 0

. 55 1 4

. 6 1 ± 0

. 50 13.4

- 22

. 0

Sum m on o

- u n s aturated 1 5

. 3 1 1 5

. 0 9 1 5

. 0 9 1 3 .9

- 2 2

. 9
**Poly**

**- u**  **n s**  **aturated**

Linoleic C 18 : 2 5 6

. 2 0 ± 1

. 71 5 7

. 3 5 ± 1

. 62 5 7

. 4 7 ± 1

. 49 36.3

- 64

. 0

Linol e n ic C 1 8 : 3 0

. 5 8 ± 0

. 14 0

. 5 6 ± 0

. 04 0

. 5 5 ± 0

. 04 < 0 .1 0

- 0

. 62

Sum pol y

- uns a turated 5 6

. 7 8 5 7

. 9 1 5 8

. 0 2 3 6 .3

- 6 4

. 3

Sum of t o tal f a tty acids 9 9

. 0 1 9 9

. 1 1 9 9

. 1 1

-

Data re pr e sent an av er age of three r ep licate samp les at six field test sites

a Refer e nce ra ng es co mp iled fr om OECD (2004) , ILSI (2007) , Bertrand et al. (2005) , Berbe rich et al. (1996),

an d N ida et al. (1996)

Table 28

. C yclopropenoid fatty acid composition in cottonseed of event T303 -3

Para m eter Based on d ry
Refere nc e

ranges a N o n

Transg e n i Transg e n ic

Not sp ra y ed Transg e n ic Sprayed

Mean ± SD Mean ± SD Mean ± SD

M al v alic acid %rel. 0

. 3 8 9 ± 0

. 0 7 6 0

. 3 6 1 ± 0

. 0 9 3 0

. 3 5 4 ± 0

. 0 8 8 0

. 17

- 1 .5

Sterculic acid %rel. 0

. 3 3 1 ± 0

. 0 4 5 0

. 3 0 6 ± 0

. 0 5 4 0

. 3 2 4 ± 0

. 0 6 5 0

. 12

- 0

. 9 2
Dihydroster c ulic acid

%rel. 0.14 9 ± 0.020 0.15 0 ± 0.015 0.14 6 ± 0.016 0.1 1 - 0.50

Data re pr e sent an av er age of three re pl i cate s amp les at six field test sites.

a Refere nce rang es compiled from OECD (2004) , ILSI (2007)

, Calhoun et al. (1995) , Be rbe r ic h et al. (1996)

, Nida et al. (1996)

, Phelps et al. (1965), an d W ozenski and Woodburn (1975)Table 29 . Mineral and tocopherol composition in cottonseed of event T303

- 3

Para m eter Based on d ry m atter

Refere nc e

ranges a N o n

Transg e n ic Transg e n ic

Not sp ra y ed Transg e n ic Sprayed

Mean ± SD Mean ± SD Mean ± SD

Calci u m % 0

. 1 5 ± 0

. 02 0

. 2 4 ± 0

. 05 0

. 2 7 ± 0

. 09 0

. 0 9

– 0.33

Ph o s ph o r u s % 0

. 6 2 ± 0

. 08 0

. 6 5 ± 0

. 07 0

. 6 6 ± 0

. 09 0

. 3 1

– 0.86

Potassium % 1

. 1 8 ± 0

. 11 1

. 2 0 ± 0

. 06 1

. 2 1 ± 0

. 07 0

. 9 6

– 1.42

M agnes i um % 0

. 3 8 ± 0

. 03 0

. 4 1 ± 0

. 03 0

. 4 1 ± 0

. 03 0

. 2 7

– 0.49

I r on % 0

. 0 06 1 ± 0

. 0 015 0

. 0 06 3 ± 0

. 0 015 0

. 0 07 2 ± 0

. 0 021 0.0023

- 0.016

Zinc m g /kg 3 0

. 2 6 ± 3

. 40 3 7

. 0 4 ± 1 2

. 8 1 3 5

. 4 5 ± 5

. 01 1 7 .8

– 6 3 .0

α T o c opherol mg/kg 1 2 8

. 39 ± 2 5.47 1 1 5

. 31 ± 1 7.57 1 2 4

. 56 ± 1 3.41 1 6

- 245

β To c o pherol m g/kg < 6 .0 0 < 6 .0 0 < 6 .0 0 0

– 10

γ To c opherol mg / kg 4 9

. 0 0 ± 1 8

. 4 0 4 9

. 2 2 ± 1 4

. 5 0 5 8

. 3 3 ± 1 3

. 6 1 16

– 27 1

δ To c o pherol m g/ k g < 6 .0 0 < 6 .0 0 < 6 .0 0 0

– 10

T o tal
Toc opherol (vitamin E) b 177. 53 ± 3 2.31 164. 79 ± 2 7.02 182. 56 ± 2 0.61 3 2
– 536

Data re pr e sent an av er age of three r ep licate samp les at six field test sites.

a Refer en ce ranges c omp iled fr om OECD (2004) , ILSI (2007)

, US CA (1982) , Cal h ou n et al. ( 1995)

, Lun dqui st (1995) and FAO/ WHO (2001)

b Vitam in E as the sum of all toc op her ols in mg /kg dm